Methods and kits for amplifying DNA

ABSTRACT

Novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic are disclosed (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, methods of nucleic acid amplification are disclosed which are robust and efficient, while reducing the appearance of side-products. In general, the methods use priming oligonucleotides that target only one sense of a target nucleic acid, a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-pro ducts. The disclosed methods minimizes or substantially eliminate the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The disclosed methods minimize or substantially eliminate this problem, thus providing enhanced levels of sensitivity.

FIELD OF THE INVENTION

This invention relates to methods, reaction mixtures, compositions andkits for producing multiple copies of a specific nucleic acid sequenceor “target sequence” which may be present either alone or as acomponent, large or small, of a homogeneous or heterogeneous mixture ofnucleic acids. The mixture of nucleic acids may be that found in asample taken for diagnostic testing, for screening of blood products,for food, water, industrial or environmental testing, for researchstudies, for the preparation of reagents or materials for otherprocesses such as cloning, or for other purposes.

The selective amplification of specific nucleic acid sequences is ofvalue in increasing the sensitivity of diagnostic and other detectionassays while maintaining specificity; increasing the sensitivity,convenience, accuracy and reliability of a variety of researchprocedures; and providing ample supplies of specific oligonucleotidesfor various purposes.

BACKGROUND OF THE INVENTION

The detection and/or quantitation of specific nucleic acid sequences isan important technique for identifying and classifying microorganisms,diagnosing infectious diseases, detecting and characterizing geneticabnormalities, identifying genetic changes associated with cancer,studying genetic susceptibility to disease, and measuring response tovarious types of treatment. Such procedures are also useful in detectingand quantitating microorganisms in foodstuffs, water, industrial andenvironmental samples, seed stocks, and other types of material wherethe presence of specific microorganisms may need to be monitored. Otherapplications are found in the forensic sciences, anthropology,archaeology, and biology where measurement of the relatedness of nucleicacid sequences has been used to identify criminal suspects, resolvepaternity disputes, construct genealogical and phylogenetic trees, andaid in classifying a variety of life forms.

A number of methods to detect and/or quantitate nucleic acid sequencesare well known in the art. These include hybridization to a labeledprobe, and various permutations of the polymerase chain reaction (PCR),coupled with hybridization to a labeled probe. See, e.g., Mullis et al.,“Process for Amplifying, Detecting and/or Cloning Nucleic AcidSequences,” U.S. Pat. No. 4,683,195; Mullis, “Process for AmplifyingNucleic Acid Sequences,” U.S. Pat. No. 4,683,202; Mullis et al.,“Process for Amplifying, Detecting and/or Cloning Nucleic AcidSequences,” U.S. Pat. No. 4,800,159; Mullis et al. (1987) Meth. Enzymol.155, 335-350; and Murakawa et al. (1988) DNA 7,287-295. The requirementof repeated cycling of reaction temperature between several differentand extreme temperatures is a disadvantage of the PCR procedure. Inorder to make PCR convenient, expensive programmable thermal cyclinginstruments are required.

Additionally, Transcription-Mediated Amplification (TMA) methods may beused to synthesize multiple copies of a target nucleic acid sequenceautocatalytically under conditions of substantially constanttemperature, ionic strength, and pH in which multiple RNA copies of thetarget sequence autocatalytically generate additional copies. See, e.g.,Kacian et al., “Nucleic Acid Sequence Amplification Methods,” U.S. Pat.No. 5,399,491, and Kacian et al, “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,824,518, the contents of each of which patentsare hereby incorporated by reference herein. TMA is useful forgenerating copies of a nucleic acid target sequence for purposes whichinclude assays to quantitate specific nucleic acid sequences inclinical, environmental, forensic and similar samples, cloning andgenerating probes. TMA is a robust and highly sensitive amplificationsystem with demonstrated efficacy. TMA overcomes many of the problemsassociated with PCR-based amplification systems. In particular,temperature cycling is not required. Other transcription-basedamplification methods are disclosed by Malek et al., “Enhanced NucleicAcid Amplification Process,” U.S. Pat. No. 5,130,238; Davey et al.,“Nucleic Acid Amplification Process,” U.S. Pat. No. 5,409,818; Davey etal., “Method for the Synthesis of Ribonucleic Acid (RNA),” U.S. Pat. No.5,466,586; Davey et al., “Nucleic Acid Amplification Process,” U.S. Pat.No. 5,554,517; Burg et al., “Selective Amplification of TargetPolynucleotide Sequences,” U.S. Pat. No. 6,090,591; and Burg et al.,“Selective Amplification of Target Polynucleotide Sequences,” U.S. Pat.No. 6,410,276.

An inherent result of highly sensitive nucleic amplification systems isthe emergence of side-products. Side-products include molecules whichmay, in some systems, interfere with the amplification reaction, therebylowering specificity. This is because limited amplification resources,including primers and enzymes needed in the formation of primerextension and transcription products are diverted to the formation ofside-products. In some situations, the appearance of side-products canalso complicate the analysis of amplicon production by various moleculartechniques.

Accordingly, there remains a need in the art for a robust nucleic acidamplification system to synthesize multiple copies of a target nucleicacid sequence autocatalytically under conditions of substantiallyconstant temperature, ionic strength, and pH which reduces theappearance of side-products, thereby increasing specificity andimproving detection and quantitation of amplification products.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods of synthesizingmultiple copies of a target sequence which are autocatalytic (i.e., ableto cycle automatically without the need to modify reaction conditionssuch as temperature, pH, or ionic strength and using the product of onecycle in the next one). In particular, the present invention disclosesmethods of nucleic acid amplification which are robust and efficient,while reducing the appearance of side-products. The methods use apriming oligonucleotide, a promoter oligonucleotide modified to preventthe initiation of DNA synthesis therefrom (e.g., includes a 3′-blockingmoiety) and, optionally, a displacer oligonucleotide, a binding moleculeand/or a 3′-blocked extender oligonucleotide, to amplify RNA or DNAmolecules in vitro. Primers used in the disclosed methods target onlyone sense of a target nucleic acid. The methods of the present inventionminimize or substantially eliminate the emergence of side-products, thusproviding a high level of specificity. Furthermore, the appearance ofside-products can complicate the analysis of the amplification reactionby various molecular detection techniques. The present inventionminimizes or substantially eliminates this problem, thus providing anenhanced level of sensitivity.

In one embodiment, the present invention is drawn to a method ofsynthesizing multiple copies of a target sequence comprising treating atarget nucleic acid which comprises an RNA target sequence with apriming oligonucleotide and a binding molecule (e.g., terminatingoligonucleotide or digestion oligonucleotide), where the primingoligonucleotide hybridizes to the 3′-end of the target sequence suchthat a primer extension reaction can be initiated therefrom, and wherethe binding molecule binds to the target nucleic acid adjacent to ornear the 5′-end of the target sequence (by “adjacent to” is meant thatthe binding molecule binds to a base of the target nucleic acid next tothe 5′-terminal base of the target sequence and fully 5′ to the targetsequence); extending the priming oligonucleotide in a primer extensionreaction with a DNA polymerase, e.g., reverse transcriptase, to give aDNA primer extension product complementary to the target sequence, wherethe primer extension product has a 3′-end which is determined by thebinding molecule, where the 3′-end of the primer extension product iscomplementary to the 5′-end of the target sequence; separating theprimer extension product from the target sequence using an enzyme whichselectively degrades the target sequence, e.g., an enzyme with an RNAseH activity; treating the primer extension product with a promoteroligonucleotide comprising first and second regions, where the firstregion hybridizes to a 3′-region of the primer extension product to forma promoter oligonucteotide:primer extension product hybrid, where thesecond region comprises a promoter for an RNA polymerase and is situated5′ to the first region, and where the promoter oligonucleotide ismodified to prevent the initiation of DNA synthesis therefrom (e.g., ablocking moiety is situated at the 3′-terminus of the promoteroligonucleotide which prevents polymerase extension); extending the3′-end of the primer extension product in the promoteroligonucleotide:primer extension product hybrid to add a sequencecomplementary to the second region of the promoter oligonucleotide; andtranscribing from the promoter oligonucleotide:primer extension producthybrid multiple RNA products complementary to the primer extensionproduct using an RNA polymerase which recognizes the promoter in thepromoter oligonucleotide and initiates transcription therefrom.According to this embodiment, the base sequences of the resulting RNAproducts are substantially identical to the base sequence of the targetsequence. In a preferred aspect of this embodiment, the activity of theDNA polymerase is substantially limited to the formation of primerextension products comprising the priming oligonucleotide. In yetanother preferred aspect of this embodiment, the formation ofside-products in the method is substantially less than if said promoteroligonucleotide was not modified to prevent the initiation of DNAsynthesis therefrom. According to yet another preferred aspect of thisembodiment, if an oligonucleotide used in the amplification reactioncomprises a promoter for an RNA polymerase, then that oligonucleotidefurther comprises a blocking moiety situated at its 3′-terminus toprevent the initiation of DNA synthesis therefrom.

A second embodiment of the present invention is drawn to a method ofsynthesizing multiple copies of a target sequence, where the methodcomprises treating a target nucleic acid comprising an RNA targetsequence with a priming oligonucleotide which hybridizes to the 3′-endof the target sequence such that a primer extension reaction can beinitiated therefrom; extending the priming oligonucleotide in a primerextension reaction with a DNA polymerase, e.g., reverse transcriptase,to give a first DNA primer extension product having an indeterminate3′-end and comprising a base region complementary to the targetsequence; separating the first primer extension product from the targetnucleic acid using an enzyme which selectively degrades that portion ofthe target nucleic acid which is complementary to the first primerextension reaction, e.g., an enzyme with an RNAse H activity; treatingthe first primer extension product with a promoter oligonucleotidecomprising first and second regions, where the first region hybridizesto a 3′-region of the first primer extension product to form a promoteroligonucleotide:first primer extension product hybrid, where the secondregion comprises a promoter for an RNA polymerase and is situated 5′ tothe first region, and where the promoter oligonucleotide is modified toprevent the initiation of DNA synthesis therefrom (e.g., a blockingmoiety is situated at the 3′-terminus of the promoter oligonucleotidewhich prevents polymerase extension); and transcribing from the promoteroligonucleotide:first primer extension product hybrid multiple first RNAproducts complementary to at least a portion of the first primerextension product using an RNA polymerase which recognizes the promoterand initiates transcription therefrom, where the base sequences of theresulting first RNA products are substantially identical to the basesequence of the target sequence. In a preferred aspect of thisembodiment, the activity of the DNA polymerase in the method issubstantially limited to the formation of primer extension productscomprising the priming oligonucleotide. In yet another preferred aspectof this embodiment, the formation of side-products in the method issubstantially less than if the promoter oligonucleotide was not modifiedto prevent the initiation of DNA synthesis therefrom. According to yetanother preferred aspect of this embodiment, if an oligonucleotide usedin the amplification reaction comprises a promoter for an RNApolymerase, then that oligonucleotide further comprises a blockingmoiety situated at its 3′-terminus to prevent the initiation of DNAsynthesis therefrom.

This embodiment is preferably drawn to the further steps of treating afirst RNA product transcribed from the promoter oligonucleotide:firstprimer extension product with the priming oligonucleotide describedabove to form a priming oligonucleotide:first RNA product hybrid suchthat a primer extension reaction can be initiated from the primingoligonucleotide; extending the priming oligonucleotide in a primerextension reaction with a DNA polymerase, e.g., reverse transcriptase,to give a second DNA primer extension product complementary to the firstRNA product, where the second primer extension product has a 3′-endwhich is complementary to the 5′-end of the first RNA product;separating the second primer extension product from the first RNAproduct using an enzyme which selectively degrades the first RNAproduct, e.g., an enzyme with an RNAse H activity; treating the secondprimer extension product with the promoter oligonucleotide describedabove to form a promoter oligonucleotide:second primer extension producthybrid; extending the 3′-end of the second primer extension product inthe promoter oligonucleotide:second primer extension product hybrid toadd a sequence complementary to the second region of the promoteroligonucleotide; and transcribing from the promoteroligonucleotide:second primer extension product hybrid multiple secondRNA products complementary to the second primer extension product usingan RNA polymerase, where the base sequences of the second RNA productsare substantially identical to the base sequence of the target sequence.

A third embodiment of the present invention is drawn to a method ofsynthesizing multiple copies of a target sequence comprising treating atarget nucleic acid comprising a DNA target sequence with a promoteroligonucleotide comprising first and second regions, where the firstregion hybridizes to the 3′-end of the target sequence to form apromoter oligonucleotide:target nucleic acid hybrid, where the secondregion comprises a promoter for an RNA polymerase and is situated 5′ tothe first region, and where the promoter oligonucleotide is modified toprevent the initiation of DNA synthesis therefrom (e.g., a blockingmoiety is situated at the 3′-terminus of the promoter oligonucleotide);transcribing from the promoter oligonucleotide:target nucleic acidhybrid multiple first RNA products comprising a base regioncomplementary to the target sequence using an RNA polymerase whichrecognizes the promoter and initiates transcription therefrom; treatingthe first RNA products with a priming oligonucleotide which hybridizesto a 3′-region of the first RNA products such that a primer extensionreaction may be initiated therefrom; extending the primingoligonucleotide in the primer extension reaction with a DNA polymerase,e.g., reverse transcriptase, to give a DNA primer extension productcomplementary to at least a portion of the first RNA products, where theprimer extension product has a 3′-end which is complementary to the5′-end of the first RNA products; separating the primer extensionproduct from the first RNA product using an enzyme which selectivelydegrades the first RNA product (e.g., an enzyme with an RNAse Hactivity); treating the primer extension product with the promoteroligonucleotide described above to form a promoteroligonucleotide:primer extension product hybrid; and transcribing fromthe promoter oligonucleotide:primer extension product hybrid multiplesecond RNA products complementary to the primer extension product usingan RNA polymerase, wherein the base sequences of the second RNA productsare substantially complementary to the base sequence of the targetsequence. In a preferred aspect of this embodiment, the activity of theDNA polymerase in the method is substantially limited to the formationof primer extension products comprising the priming oligonucleotide. Inyet another preferred aspect of this embodiment, the formation ofside-products in the method is substantially less than if the promoteroligonucleotide was not modified to prevent the initiation of DNAsynthesis therefrom. According to yet another preferred aspect of thisembodiment, if an oligonucleotide used in the amplification reactioncomprises a promoter for an RNA polymerase, then that oligonucleotidefurther comprises a blocking moiety situated at its 3′-terminus toprevent the initiation of DNA synthesis therefrom. Furthermore, anymethod of this embodiment may include extending the 3′-end of the primerextension product in the promoter oligonucleotide:primer extensionproduct hybrid described above to add a sequence complementary to thesecond region of the promoter oligonucleotide.

A fourth embodiment of the present invention is drawn to a method ofsynthesizing multiple copies of a target sequence comprising treating atarget nucleic acid which comprises a DNA target sequence with a primingoligonucleotide, where the priming oligonucleotide hybridizes to the3′-end of the target sequence such that a primer extension reaction canbe initiated therefrom; extending the priming oligonucleotide in aprimer extension reaction with a DNA polymerase, e.g., reversetranscriptase, to give a first DNA primer extension product, where atleast a portion of the first primer extension product is complementaryto the target sequence; treating the primer extension product with apromoter oligonucleotide comprising first and second regions, where thefirst region comprises a base sequence which corresponds to a region atthe 5′-end of the target sequence and which hybridizes to the firstprimer extension product to form a promoter oligonucleotide:first primerextension product hybrid, where the second region comprises a promoterfor an RNA polymerase and is situated 5′ to the first region, and wherethe promoter oligonucleotide is modified to prevent the initiation ofDNA synthesis therefrom (e.g., a blocking moiety is situated at the3′-terminus of the promoter oligonucleotide which prevents polymeraseextension); and transcribing from the promoter oligonucleotide:firstprimer extension product hybrid multiple first RNA productscomplementary to at least a portion of the first primer extensionproduct using an RNA polymerase which recognizes the promoter in thepromoter oligonucleotide and initiates transcription therefrom. Providedthat if the first primer extension product has a defined 3′-end, thenthe method further comprises treating the target nucleic acid with abinding molecule which binds to the target nucleic acid adjacent to ornear the 5′-end of the target sequence. Further provided that thepriming oligonucleotide does not include an RNA region which hybridizesto the target nucleic acid and which is selectively degraded by anenzyme activity when hybridized to the target nucleic acid. According tothis embodiment, the base sequences of the resulting first RNA productsare substantially identical to the base sequence of the target sequence.In a preferred aspect of this embodiment, the target nucleic acid ispart of a double-stranded complex that is exposed to conditionssufficient to denature the complex (e.g., heat and/or chemicaldenaturants) prior to extending the priming oligonucleotide in a primerextension reaction. In another preferred aspect of this embodiment, theactivity of the DNA polymerase is substantially limited to the formationof primer extension products comprising the priming oligonucleotide. Inyet another preferred aspect of this embodiment, the formation ofside-products in the method is substantially less than if said promoteroligonucleotide was not modified to prevent the initiation of DNAsynthesis therefrom. In yet a further preferred aspect of thisembodiment, if an oligonucleotide used in the amplification reactioncomprises a promoter for an RNA polymerase, then that oligonucleotidefurther comprises a blocking moiety situated at its 3′-terminus toprevent the initiation of DNA synthesis therefrom.

The method of this embodiment is preferably drawn to the further stepsof treating the first RNA products transcribed from the promoteroligonucleotide:first DNA primer extension product with the primingoligonucleotide described above to form a priming oligonucleotide:firstRNA product hybrid such that a primer extension reaction can beinitiated from the priming oligonucleotide; extending the primingoligonucleotide in a primer extension reaction with a DNA polymerase,e.g., reverse transcriptase, to give a second DNA primer extensionproduct complementary to the first RNA product, where the second primerextension product has a 3′-end which is complementary to the 5′-end ofthe first RNA product; separating the second primer extension productfrom the first RNA product using an enzyme which selectively degradesthe first RNA product, e.g., an enzyme with an RNAse H activity;treating the second primer extension product with the promoteroligonucleotide described above to form a promoteroligonucleotide:second primer extension product hybrid; extending the3′-end of the second primer extension product in the promoteroligonucleotide:second primer extension product hybrid to add a sequencecomplementary to the second region of the promoter oligonucleotide; andtranscribing from the promoter oligonucleotide:second primer extensionproduct hybrid multiple second RNA products complementary to the secondprimer extension product using an RNA polymerase, where the basesequences of the second RNA products are substantially identical to thebase sequence of the target sequence.

Another aspect of the method of this embodiment comprises treating thetarget nucleic acid with a binding molecule (e.g., terminatingoligonucleotide or digestion oligonucleotide), where the bindingmolecule binds to the target nucleic acid adjacent to or near the 5′-endof the target sequence (as indicated above, the phrase “adjacent to”means that the binding molecule binds to a base sequence of the targetnucleic acid next to the 5′-terminal base of the target sequence andfully 5′ to the target sequence). The target nucleic acid is preferablytreated with the binding molecule prior to initiating extension of thepriming oligonucleotide in a primer extension reaction with a DNApolymerase. In this aspect, the first primer extension product has a3′-end which is determined by the binding molecule, where the 3′-end ofthe primer extension product is complementary to the 5′-end of thetarget sequence. After the promoter oligonucleotides hybridizes to thefirst primer extension product, this aspect of the method furthercomprises extending the 3′-end of the first DNA primer extension productin the promoter oligonucleotide:first primer extension product hybrid toadd a sequence complementary to the second region of the promoteroligonucleotide.

Yet another aspect of the method of this embodiment comprises treatingthe target nucleic acid with a displacer oligonucleotide, where thedisplacer oligonucleotide hybridizes to the target nucleic acid upstreamfrom the priming oligonucleotide such that a primer extension reactioncan be initiated therefrom (in this context, the term “upstream” meansthat the 3′-terminal nucleotide of the displacer oligonucleotide is 5′to the 3′-terminal nucleotide of the priming oligonucleotide), and thenextending the displacer oligonucleotide in a primer extension reactionwith a DNA polymerase to give a second DNA primer extension product thatdisplaces the first DNA primer extension product from the target nucleicacid. In a preferred aspect, the activity of the DNA polymerase issubstantially limited to the formation of primer extension productscomprising the displacer and priming oligonucleotides.

Reagents and conditions suitable for practicing any of the embodimentsdescribed above are set forth in the Examples section.

The methods of the present invention may be used as a component ofassays to detect and/or quantitate specific nucleic acid targetsequences in clinical, food, water, industrial, environmental, forensic,and similar samples or to produce large numbers of copies of DNA and/orRNA of specific target sequences for a variety of uses. (As used herein,the term “copies” refers to amplification products having either thesame or the opposite sense of the target sequence.) These methods mayalso be used to produce multiple copies of a target sequence for cloningor to generate probes or to produce RNA and DNA copies for sequencing.

The priming oligonucleotides of the embodiments described aboveoptionally have a cap comprising a base region hybridized to a 3′-endthereof prior to treating a target nucleic acid or an RNA product withone of the priming oligonucleotides in order to prevent the initiationof DNA synthesis therefrom. (As used herein, the term “primingoligonucleotide” is inclusive of displacer oligonucleotides.) The5′-terminal base (i.e., the 5′-most base) of a cap hybridizes to the3′-terminal base (i.e., the 3′-most base) of a priming oligonucleotide.However, the caps are designed to be preferentially displaced frompriming oligonucleotides by a target nucleic acid, a primer extensionproduct, or an RNA product. A cap of the present invention may take theform of a discrete capping oligonucleotide, or may be attached to the5′-end of a priming oligonucleotide via a linker. A preferred cappingoligonucleotide is modified to prevent the initiation of DNA synthesistherefrom (e.g., comprises a blocking moiety at its 3′-terminus).

To increase the binding affinity of a priming oligonucleotide for atarget nucleic acid or complement thereof, the 5′-end of a primingoligonucleotide may include one or more modifications which improve thebinding properties (e.g., hybridization or base stacking) of the primingoligonucleotide to the target nucleic acid or an RNA product, providedthe modifications do not prevent the priming oligonucleotide from beingextended in a primer extension reaction or substantially interfere withcleavage of an RNA template to which the priming oligonucleotide ishybridized. The modifications are preferably spaced at least 15 basesfrom the 3′-terminus of a priming oligonucleotide, and most preferablyaffect a region limited to the three or four 5′-most nucleotides of thepriming oligonucleotide. Preferred modifications include 2′-O-methylribonucleotides and “Locked Nucleic Acids” or “Locked NucleosideAnalogues” (LNAs). See Becker et al., “Method for Amplifying TargetNucleic Acids Using Modified Primers,” U.S. Pat. No. 613,038; Imanishiet al, “Bicyclonucleoside and Oligonucleotide Analogues,” U.S. Pat. No.6,268,490; and Wengel et al., “Oligonucleotide Analogues,” U.S. Pat. No.6,670,461. The contents of each of the foregoing references are herebyincorporated by reference herein.

The promoter oligonucleotide used in the methods described above mayfurther include an insertion sequence which is selected to enhance therate at which RNA products are formed. The insertion sequence ispreferably from 5 to 20 nucleotides in length and is positioned betweenor adjacent to the first and second regions of the promoteroligonucleotide. Preferred insertion sequences of the present inventioninclude the base sequences of SEQ ID NO:1 ccacaa and SEQ ID NO:2acgtagcatcc.

The rate of amplification may also be affected by the inclusion of anextender oligonucleotide in any of the above-described methods. Anextender oligonucleotide is preferably from 10 to 50 nucleotides inlength and is designed to hybridize to a DNA template so that the 5′-endof the extender oligonucleotide is adjacent to or near the 3′-end of apromoter oligonucleotide. The extender oligonucleotide is preferablymodified to prevent the initiation of DNA synthesis therefrom (e.g.,includes a 3′-terminal blocking moiety).

In some applications of the methods described above, the bindingmolecule may comprise an oligonucleotide having a 5′-end which overlapsthe 5′-end of the first region of the promoter oligonucleotide. To limithybridization of the binding molecule to the promoter oligonucleotide,the 5′-end of the first region of the promoter oligonucleotide may besynthesized to include a sufficient number of mismatches with the 5′-endof the binding molecule to prevent the promoter oligonucleotide fromhybridizing to the binding molecule. While a single mismatch generallyshould be sufficient, the number of destabilizing mismatches needed inthe first region of the promoter oligonucleotide will depend upon thelength and base composition of the overlapping region.

In an adaptation of the above methods, the blocking moiety may bereleased from the promoter oligonucleotide prior to treating the primerextension product or the first primer extension product with thepromoter oligonucleotide. To facilitate release of the blocking moiety,the promoter oligonucleotide is provided to a reaction mixturepre-hybridized to an oligodeoxynucleotide. The oligodeoxynucleotide ishybridized to a 3′-region of the first region of the promoteroligonucleotide which includes a sufficient number of contiguousribonucleotides such that the blocking moiety is released from thepromoter oligonucleotide in the presence of an enzymatic activitycapable of cleaving the ribonucleotides of the 3′-region. Duringcleavage of the ribonucleotides, the oligodeoxynucleotide is alsoreleased from the first region of the promoter oligonucleotide, and theremaining, uncleaved portion of the first region hybridizes to theprimer extension product or the first primer extension product. The3′-section of ribonucleotides preferably includes at least 6 contiguousribonucleotides, and the oligodeoxynucleotide is preferably the samelength as and fully complementary to the 3′-section of ribonucleotides.The oligodeoxynucleotide may be a separate molecule or it may be joinedto the promoter oligonucleotide by means of a linker.

The present invention further relates to reaction mixtures useful forcarrying out the methods described above. The reaction mixtures of thepresent invention may contain each component, or some subcombination ofcomponents, necessary for carrying out the methods described above.

The materials and/or reagents used in the methods of the presentinvention may be incorporated as parts of kits, e.g., diagnostic kitsfor clinical or criminal laboratories, or nucleic amplification kits forgeneral laboratory use. The present invention thus includes kits whichinclude some or all of the components necessary to carry out the methodsof the present invention, e.g., oligonucleotides, binding molecules,stock solutions, enzymes, positive and negative control targetsequences, detection reagents, containers (e.g., test tubes, cuvettes,cassettes, plates, microfluidic devices and the like), and instructionsprovided in written or electronic form for performing the disclosedmethods.

Certain embodiments of the present invention include one or moredetection probes for determining the presence or amount of the RNAand/or DNA products in the amplification reaction mixture. Probes may bedesigned to detect RNA and/or DNA products after the amplificationreaction (i.e., end-point detection) or, alternatively, during theamplification reaction (i.e., real-time detection involves periodicallymeasuring the amount of signal associated with probe:amplicon complexesin the reaction mixture). Thus, the probes may be provided to thereaction mixture prior to, during or at the completion of theamplification reaction. For real-time detection of RNA products in thefirst two methods described above, it may be desirable to provide theprobe to the reaction mixture after the first primer extension reactionhas been initiated (i.e., addition of amplification enzymes) since probebinding to the target sequence, rather than RNA product, may slow therate at which an RNA-dependent DNA polymerase (e.g., reversetranscriptase) can extend the priming oligonucleotide. Preferred probeshave one or more associated labels to facilitate detection.

The present invention is further drawn to various oligonucleotides,including the priming oligonucleotides, promoter oligonucleotides,terminating oligonucleotides, displacer oligonucleotides, cappingoligonucleotides, extender oligonucleotides and detection probesdescribed herein. It is to be understood that oligonucleotides of thepresent invention may be DNA or RNA (and analogs thereof), and in eithercase, the present invention includes RNA equivalents of DNAoligonucleotides and DNA equivalents of RNA oligonucleotides. Except forthe preferred priming oligonucleotides, displacer oligonucleotides anddetection probes described below, the oligonucleotides described in thefollowing paragraphs are preferably modified to prevent theirparticipation in a synthesis reaction in the presence of a DNApolymerase (e.g., include a blocking moiety at their 3′-termini).

For certain amplification reactions in which the target nucleic acidcontains a hepatitis C virus (HCV) 5′ untranslated region, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3 aatttaatacgactcac tatagggaga. Thehybridizing sequence of the preferred promoter oligonucleotidecomprises, consists of, consists essentially of, overlaps with, or iscontained within and includes at least 10, 15, 20, 25, 30 or 32contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:4ctagccatggcgttagtatgagtgtcgtgcag or an equivalent sequence containinguracil bases substituted for thymine bases, and which hybridizes to thetarget nucleic acid under amplification conditions. The promoteroligonucleotide preferably does not include a region in addition to thehybridizing sequence that hybridizes to the target nucleic acid underamplification conditions. More preferably, the promoter oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:5aatttaatacgactcactatagggagactagccatggcgttagtatgagtgtcgtgcag or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. The base sequence of the promoteroligonucleotide preferably consists of a promoter sequence and ahybridizing sequence consisting of or contained within and including atleast 10, 15, 20, 25, 30 or 32 contiguous bases of the base sequence ofSEQ ID NO:4 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains an HCV 5′ untranslated region, the present invention includes apriming oligonucleotide up to 40 or 50 bases in length. A preferredpriming oligonucleotide includes an oligonucleotide comprising,consisting of, consisting essentially of, overlapping with, or containedwithin and including at least 10, 15, 20, 25, 30 or 31 contiguous basesof a base sequence that is at least 80%, 90% or 100% identical to thebase sequence of SEQ ID NO:6 aggcattgagcgggttgatccaagaaaggac or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. More preferably, the priming oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:6 or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes under amplification conditions to the targetnucleic acid. The base sequence of the priming oligonucleotidepreferably consists of or is contained within and includes at least 10,15, 20, 25, 30 or 31 contiguous bases of the base sequence of SEQ IDNO:6 or an equivalent sequence containing uracil bases substituted forthymine bases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains an HCV 5′ untranslated region, the present invention is furtherdirected to a detection probe up to 35, 50 or 100 bases in length. Apreferred detection probe includes a target binding region whichcomprises, consists of, consists essentially of, overlaps with, or iscontained within and includes at least 10, 13 or 15 contiguous bases ofa base sequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:7 guacucaccgguucc, the complement thereof, or anequivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement (e.g., not human nucleic acid) under stringenthybridization conditions. The detection probe preferably does notinclude a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:7, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 10, 13 or 15 contiguousbases of the base sequence of SEQ ID NO:7, the complement thereof, or anequivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains an HCV 5′ untranslated region, the present invention is furtherdirected to a detection probe up to 40 or 50 bases in length. Apreferred detection probe includes a target binding region whichcomprises, consists of, consists essentially of, overlaps with, or iscontained within and includes at least 18, 20 or 22 contiguous bases ofa base sequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:8 agaccacuauggcucucccggg, the complement thereof,or an equivalent sequence containing thymine bases substituted foruracil bases, and which preferentially hybridizes to the target nucleicacid or its complement (e.g., not human nucleic acid) under stringenthybridization conditions. The detection probe preferably does notinclude a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:8, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 18, 20 or 22 contiguousbases of the base sequence SEQ ID NO:8, the complement thereof, or anequivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a human immunodeficiency virus (HIV) pol gene, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25, 30 or 31 contiguous bases of a base sequence thatis at least 80%, 90% or 100% identical to the base sequence of SEQ IDNO:9 acaaatggcagtattcatccacaatttaaaa or an equivalent sequencecontaining uracil bases substituted for thymine bases, and whichhybridizes to the target nucleic acid under amplification conditions.The promoter oligonucleotide preferably does not include a region inaddition to the hybridizing sequence that hybridizes to the targetnucleic acid under amplification conditions. More preferably, thepromoter oligonucleotide comprises, consists of, or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:10 aatttaatacgactcactatagggagactagccatggcgttagtatgagtgtcgtgcag or an equivalent sequence containinguracil bases substituted for thymine bases, and which hybridizes to thetarget nucleic acid under amplification conditions. The base sequence ofthe promoter oligonucleotide preferably consists of a promoter sequenceand a hybridizing sequence consisting of or contained within andincluding at least 10, 15, 20, 25, 30 or 31 contiguous bases of the basesequence of SEQ ID NO:9 or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains an HIV pot gene, the present invention includes a primingoligonucleotide up to 40 or 50 bases in length. A preferred primingoligonucleotide includes an oligonucleotide comprising, consisting of,consisting essentially of, overlapping with, or contained within andincluding at least 10, 15, 20, 25 or 27 contiguous bases of a basesequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:11 gtttgtatgtctgttgctattatgtct or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. More preferably, the priming oligonucleotide comprises,consists of, or consists essentially of a base sequence substantiallycorresponding to the base sequence of SEQ ID NO:11 or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. The base sequence of the priming oligonucleotide preferablyconsists of or is contained within and includes at least 10, 15, 20, 25or 27 contiguous bases of the base sequence of SEQ ID NO:11 or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains an HIV pol gene, the present invention is further directed to adetection probe up to 35, 50 or 100 bases in length. A preferreddetection probe includes a target binding region which comprises,consists of, consists essentially of, overlaps with, or is containedwithin and includes at least 13, 15 or 17 contiguous bases of a basesequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:12 acuguaccccccaaucc, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement (e.g., not human nucleic acid) under stringenthybridization conditions. The detection probe preferably does notinclude a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:12, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 13, 15 or 17 contiguousbases of the base sequence of SEQ ID NO:12, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes under stringent hybridizationconditions to the target nucleic acid or its complement. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a human papilloma virus (HPV) E6 and E7 gene, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25 or 27 contiguous bases of a base sequence that isat least 80%, 90% or 100% identical to the base sequence of SEQ ID NO:13gaacagatggggcacacaattcctagt or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The promoteroligonucleotide preferably does not include a region in addition to thehybridizing sequence that hybridizes to the target nucleic acid underamplification conditions. More preferably, the promoter oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:14aatttaatacgactcactatagggagagaa cagatggggcacacaattcctagt or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. The base sequence of the promoter oligonucleotide preferablyconsists of a promoter sequence and a hybridizing sequence consisting ofor contained within and including at least 10, 15, 20, 25 or 27contiguous bases of the base sequence of SEQ ID NO:13 or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions.

For certain amplification reactions in which the target nucleic acidcontains an HPV E6 and E7 gene, the present invention includes a primingoligonucleotide up to 40 or 50 bases in length. A preferred primingoligonucleotide includes an oligonucleotide comprising, consisting of,consisting essentially of, overlapping with, or contained within andincluding at least 10, 15 or 19 contiguous bases of a base sequence thatis at least 80%, 90% or 100% identical to the base sequence of SEQ IDNO:15 gacagctcagaggaggagg or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. More preferably, thepriming oligonucleotide comprises, consists of; or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:15 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The base sequence of thepriming oligonucleotide preferably consists of or is contained withinand includes at least 10, 15 or 19 contiguous bases of the base sequenceof SEQ ID NO:15 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains an HPV E6 and E7 gene, the present invention is furtherdirected to a detection probe up to 35, 50 or 100 bases in length, Apreferred detection probe includes a target binding region whichcomprises, consists of, consists essentially of, overlaps with, or iscontained within and includes at least 15, 17 or 19 contiguous bases ofa base sequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:16 ggacaagcagaaccggaca or the complement thereof,and which preferentially hybridizes to the target nucleic acid or itscomplement (e.g., not human nucleic acid) under stringent hybridizationconditions. The detection probe preferably does not include a region inaddition to the target binding region that hybridizes to the targetnucleic acid or its complement under stringent hybridization conditions.More preferably, the detection probe comprises, consists of, or consistsessentially of a base sequence substantially corresponding to the basesequence of SEQ ID NO:16 or the complement thereof, and whichpreferentially hybridizes to the target nucleic acid or its complementunder stringent hybridization conditions. The base sequence of thedetection probe preferably consists of or is contained within andincludes at least 15, 17 or 19 contiguous bases of the base sequence ofSEQ ID NO:16 or the complement thereof, and which preferentiallyhybridizes to the target nucleic acid or its complement under stringenthybridization conditions. In certain embodiments the probe optionallyincludes one or more detectable labels, e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a West Nile Virus (WNV) nonstructural protein 5 gene, thepresent invention includes a promoter oligonucleotide comprising apromoter sequence and a hybridizing sequence up to 40 or 50 bases inlength. The promoter sequence is recognized by an RNA polymerase, suchas a T7, T3 or SP6 RNA polymerase, and preferably includes the T7 RNApolymerase promoter sequence of SEQ ID NO:3. The hybridizing sequence ofthe preferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25 or 27 contiguous bases of a base sequence that isat least 80%, 90% or 100% identical to the base sequence of SEQ ID NO:17gagtagacggtgctgcctgcgactcaa or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The promoteroligonucleotide preferably does not include a region in addition to thehybridizing sequence that hybridizes to the target nucleic acid underamplification conditions. More preferably, the promoter oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:18aatttaatacgactcactcactatagggagagagtagacggtgctgcctgcgactcaa or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. The base sequence of the promoteroligonucleotide preferably consists of a promoter sequence and ahybridizing sequence consisting of or contained within and including atleast 10, 15, 20, 25 or 27 contiguous bases of the base sequence of SEQID NO:17 or an equivalent sequence containing uracil bases substitutedfor thymine bases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a WNV nonstructural protein 5 gene, the present inventionincludes a priming oligonucleotide up to 40 or 50 bases in length. Apreferred priming oligonucleotide includes an oligonucleotidecomprising, consisting of, consisting essentially of, overlapping with,or contained within and including at least 10, 15, 20 or 23 contiguousbases of a base sequence that is at least 80%, 90% or 100% identical tothe base sequence of SEQ ID NO:19 tccgagacggttctgagggctta or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. More preferably, the priming oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:19 or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. The base sequence of the primingoligonucleotide preferably consists of or is contained within andincludes at least 10, 15, 20 or 23 contiguous bases of the base sequenceof SEQ ID NO:19 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a WNV nonstructural protein 5 gene, the present invention isfurther directed to a detection probe up to 35, 50 or 100 bases inlength. A preferred detection probe includes a target binding regionwhich comprises, consists of, consists essentially of, overlaps with, oris contained within and includes at least 14, 16 or 18 contiguous basesof a base sequence that is at least 80%, 90% or 100% identical to thebase sequence of SEQ ID NO:20 gaucacuucgcggcuuug, the complementthereof, or an equivalent sequence containing thymine bases substitutedfor uracil bases, and which preferentially hybridizes to the targetnucleic acid or its complement (e.g., not human nucleic acid) understringent hybridization conditions. The detection probe preferably doesnot include a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:20, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 14, 16 or 18 contiguousbases of the base sequence of SEQ ID NO:20, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a 23S rRNA sequence of Chlamydia trachomatis, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25 or 30 contiguous bases of a base sequence that isat least 80%, 90% or 100% identical to the base sequence of SEQ ID NO:21cggagtaagttaagcacgcggacgattgga or an equivalent sequence containinguracil bases substituted for thymine bases, and which hybridizes to thetarget nucleic acid under amplification conditions. The promoteroligonucleotide preferably does not include a region in addition to thehybridizing sequence that hybridizes to the target nucleic acid underamplification conditions. More preferably, the promoter oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:22aatttaatacgactcactatagggagacgg agtaagttaagcacgcggacgattgga or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. The base sequence of the promoteroligonucleotide preferably consists of a promoter sequence and ahybridizing sequence consisting of or contained within and including atleast 10, 15, 20, 25 or 30 contiguous bases of the base sequence of SEQID NO:21 or an equivalent sequence containing uracil bases substitutedfor thymine bases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a 23S rRNA sequence of Chlamydia trachomatis, the presentinvention includes a priming oligonucleotide up to 40 or 50 bases inlength. A preferred priming oligonucleotide includes an oligonucleotidecomprising, consisting of, consisting essentially of, overlapping with,or contained within and including at least 10, 15, 20, 25 or 29contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:23cccgaagattccccttgatcgcgacctga or an equivalent sequence containinguracil bases substituted for thymine bases, and which hybridizes to thetarget nucleic acid under amplification conditions. More preferably, thepriming oligonucleotide comprises, consists of, or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:23 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The base sequence of thepriming oligonucleotide preferably consists of or is contained withinand includes at least 10, 15, 20, 25 or 29 contiguous bases of the basesequence of SEQ ID NO:23 or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a 23S rRNA sequence of Chlamydia trachomatis, the presentinvention is further directed to a detection probe up to 35, 50 or 100bases in length. A preferred detection probe includes a target bindingregion which comprises, consists of, consists essentially of, overlapswith, or is contained within and includes at least 19, 22 or 24contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:24 cguucucaucgcucuacggacucu,the complement thereof or an equivalent sequence containing thyminebases substituted for uracil bases, and which preferentially hybridizesto the target nucleic acid or its complement (e.g., not Chlamydiapsittaci nucleic acid) under stringent hybridization conditions. Thedetection probe preferably does not include a region in addition to thetarget binding region that hybridizes to the target nucleic acid or itscomplement under stringent hybridization conditions. More preferably,the detection probe comprises, consists of, or consists essentially of abase sequence substantially corresponding to the base sequence of SEQ IDNO:24, the complement thereof, or an equivalent sequence containingthymine bases substituted for uracil bases, and which preferentiallyhybridizes under stringent hybridization conditions to the targetnucleic acid or its complement. The base sequence of the detection probepreferably consists of or is contained within and includes at least 19,22 or 24 contiguous bases of the base sequence of SEQ ID NO:24, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. In certain embodiments the probe optionally includes one ormore detectable labels, e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25, 30, 35 or 36 contiguous bases of a base sequencethat is at least 80%, 90% or 100% identical to the base sequence of SEQID NO:25 actgggtetaataccggataggaccacgggatgcat or an equivalent sequencecontaining uracil bases substituted for thymine bases, and whichhybridizes to the target nucleic acid under amplification conditions.The promoter oligonucleotide preferably does not include a region inaddition to the hybridizing sequence that hybridizes to the targetnucleic acid under amplification conditions. More preferably, thepromoter oligonucleotide comprises, consists of, or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:26aattctaatacgactcactatagggagaactgggtctaataccggataggaccacgggatgcat or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions. The base sequence of the promoteroligonucleotide preferably consists of a promoter sequence and ahybridizing sequence consisting of or contained within and including atleast 10, 15, 20, 25, 30, 35 or 36 contiguous bases of the base sequenceof SEQ ID NO:25 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention includes a promoter oligonucleotide comprising a promotersequence and a hybridizing sequence up to 40 or 50 bases in length. Thepromoter sequence is recognized by an RNA polymerase, such as a T7, T3or SP6 RNA polymerase, and preferably includes the T7 RNA polymerasepromoter sequence of SEQ ID NO:3. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25, 30 or 31 contiguous bases of a base sequence thatis at least 80%, 90% or 100% identical to the base sequence of SEQ IDNO:27 actgggtctaataccggataggaccacggga or an equivalent sequencecontaining uracil bases substituted for thymine bases, and whichhybridizes to the target nucleic acid under amplification conditions.The promoter oligonucleotide preferably does not include a region inaddition to the hybridizing sequence that hybridizes to the targetnucleic acid under amplification conditions. More preferably, thepromoter oligonucleotide comprises, consists of, or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:28 aattctaatacgactcactatagggagaactgggtctaataccggataggaccacggga or an equivalent sequencecontaining uracil bases substituted for thymine bases, and whichhybridizes to the target nucleic acid under amplification conditions.The base sequence of the promoter oligonucleotide preferably consists ofa promoter sequence and a hybridizing sequence consisting of orcontained within and including at least 10, 15, 20, 25, 30 or 31contiguous bases of the base sequence of SEQ ID NO:27 or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention includes a priming oligonucleotide up to 40 or 50 bases inlength. A preferred priming oligonucleotide includes an oligonucleotidecomprising, consisting of, consisting essentially of, overlapping with,or contained within and including at least 10, 15, 20, 25 or 27contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:29gccgtcaccccaccaacaagctgatag or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. More preferably, thepriming oligonucleotide comprises, consists of, or consists essentiallyof a base sequence substantially corresponding to the base sequence ofSEQ ID NO:29 or an equivalent sequence containing uracil basessubstituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The base sequence of thepriming oligonucleotide preferably consists of or is contained withinand includes at least 10, 15, 20, 25 or 27 contiguous bases of the basesequence of SEQ ID NO:29 or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention is further directed to a detection probe up to 35, 50 or 100bases in length. A preferred detection probe includes a target bindingregion which comprises, consists of, consists essentially of, overlapswith, or is contained within and includes at least 18, 20 or 22contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:30 gcucaucceacaccgcuaaagc,the complement thereof, or an equivalent sequence containing thyminebases substituted for uracil bases, and which preferentially hybridizesto the target nucleic acid or its complement (e.g., not nucleic acidfrom a Mycobacterium avium complex organism) under stringenthybridization conditions. The detection probe preferably does notinclude a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:30, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 18, 20 or 22 contiguousbases of the base sequence of SEQ ID NO:30, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention is further directed to a detection probe up to 35, 50 or 100bases in length. A preferred detection probe includes a target bindingregion which comprises, consists of, consists essentially of, overlapswith, or is contained within and includes at least 22, 25 or 28contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:31ccgagaucccacaccgcuaaagccucgg, the complement thereof, or an equivalentsequence containing thymine bases substituted for uracil bases, andwhich preferentially hybridizes to the target nucleic acid or itscomplement (e.g., not nucleic acid from a Mycobacterium avium complexorganism) under stringent hybridization conditions. The detection probepreferably does not include a region in addition to the target bindingregion that hybridizes to the target nucleic acid or its complementunder stringent hybridization conditions. More preferably, the detectionprobe comprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:31, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 22, 25 or 28 contiguousbases of the base sequence of SEQ ID NO:31, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., a fluorophore/quencher dye pair.

For certain amplification reactions in which the target nucleic acidcontains a 16S rRNA sequence of Mycobacterium tuberculosis, the presentinvention is further directed to a detection probe up to 35, 50 or 100bases in length. A preferred detection probe includes a target bindingregion which comprises, consists of, consists essentially of, overlapswith, or is contained within and includes at least 18, 20 or 22contiguous bases of a base sequence that is at least 80%, 90% or 100%identical to the base sequence of SEQ ID NO:32 gctcatcccacaccgctaaagc,the complement thereof, or an equivalent sequence containing uracilbases substituted for thymine bases, and which preferentially hybridizesto the target nucleic acid or its complement (e.g., not nucleic acidfrom a Mycobacterium avium complex organism) under stringenthybridization conditions. The detection probe preferably does notinclude a region in addition to the target binding region thathybridizes to the target nucleic acid or its complement under stringenthybridization conditions. More preferably, the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:32, thecomplement thereof, or an equivalent sequence containing uracil basessubstituted for thymine bases, and which preferentially hybridizes tothe target nucleic acid or its complement under stringent hybridizationconditions. The base sequence of the detection probe preferably consistsof or is contained within and includes at least 18, 20 or 22 contiguousbases of the base sequence of SEQ ID NO:32, the complement thereof, oran equivalent sequence containing uracil bases substituted for thyminebases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. In certainembodiments the probe optionally includes one or more detectable labels,e.g., an AE substituent.

For certain amplification reactions in which the target nucleic acidcontains the orfX gene of a Staphylococcus aureus (e.g. amethicillin-resistant strain), the present invention includes a promoteroligonucleotide comprising a promoter sequence and a hybridizingsequence up to 40 or 50 bases in length. The promoter sequence isrecognized by an RNA polymerase, such as a T7, T3 or SP6 RNA polymerase,and preferably includes the T7 RNA polymerase promoter sequence of SEQID NO:3 aatttaatacgactcactatagggaga. The hybridizing sequence of thepreferred promoter oligonucleotide comprises, consists of, consistsessentially of, overlaps with, or is contained within and includes atleast 10, 15, 20, 25 or 29 contiguous bases of a base sequence that isat least 80%, 90% or 100% identical to the base sequence of SEQ ID NO:33tgacccaagggcaaagcgactttg or an equivalent sequence containing uracilbases substituted for thymine bases, and which hybridizes to the targetnucleic acid under amplification conditions. The promoteroligonucleotide preferably does not include a region in addition to thehybridizing sequence that hybridizes to the target nucleic acid underamplification conditions. More preferably, the promoter oligonucleotidecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:34aatttaatacgactcactatagggagatgacccaagggcaaagcgactttg or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. The base sequence of the promoter oligonucleotide preferablyconsists of a promoter sequence, such as SEQ ID NO:3, and a hybridizingsequence consisting of or contained within and including at least 10,15, 20, 25 or 29 contiguous bases of the base sequence of SEQ ID NO:33or an equivalent sequence containing uracil bases substituted forthymine bases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains the orfX gene of a Staphylococcus aureus (e.g. amethicillin-resistant strain), the present invention includes a primingoligonucleotide up to 40 or 50 bases in length. A preferred primingoligonucleotide includes an oligonucleotide comprising, consisting of,consisting essentially of, overlapping with, or contained within andincluding at least 10, 15, 20, 25 or 26 contiguous bases of a basesequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:35 gtgcgtagttactgcgttgtaagacgtc or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. More preferably, the priming oligonucleotide comprises,consists of, or consists essentially of a base sequence substantiallycorresponding to the base sequence of SEQ ID NO:35 or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes under amplification conditions to the target nucleicacid. The base sequence of the priming oligonucleotide preferablyconsists of or is contained within and includes at least 10, 15, 20, 25or 26 contiguous bases of the base sequence of SEQ ID NO:35 or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains the orfX gene of a Staphylococcus aureus (e.g. amethicillin-resistant strain), the present invention includes a primingoligonucleotide up to 40 or 50 bases in length. A preferred primingoligonucleotide includes an oligonucleotide comprising, consisting of,consisting essentially of, overlapping with, or contained within andincluding at least 10, 15, 20, 25 or 26 contiguous bases of a basesequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:36 ctgaatgatagtgcgtagttactgcg or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes to the target nucleic acid under amplificationconditions. More preferably, the priming oligonucleotide comprises,consists of, or consists essentially of a base sequence substantiallycorresponding to the base sequence of SEQ ID NO:36 or an equivalentsequence containing uracil bases substituted for thymine bases, andwhich hybridizes under amplification conditions to the target nucleicacid. The base sequence of the priming oligonucleotide preferablyconsists of or is contained within and includes at least 10, 15, 20, 25or 26 contiguous bases of the base sequence of SEQ ID NO:36 or anequivalent sequence containing uracil bases substituted for thyminebases, and which hybridizes to the target nucleic acid underamplification conditions.

For certain amplification reactions in which the target nucleic acidcontains the orfX gene of a Staphylococcus aureus (e.g. amethicillin-resistant strain), the present invention is further directedto a detection probe up to 35, 50 or 100 bases in length. A preferreddetection probe includes a target binding region which comprises,consists of, consists essentially of, overlaps with, or is containedwithin and includes at least 10, 12, 15 or 17 contiguous bases of a basesequence that is at least 80%, 90% or 100% identical to the basesequence of SEQ ID NO:37 ccgucauuggcggauca, the complement thereof, oran equivalent sequence containing thymine bases substituted for uracilbases, and which preferentially hybridizes to the target nucleic acid orits complement under stringent hybridization conditions. The detectionprobe preferably does not include a region in addition to the targetbinding region that hybridizes to the target nucleic acid or itscomplement under stringent hybridization conditions. More preferably,the base sequence of the target binding region of the detection probecomprises, consists of, or consists essentially of a base sequencesubstantially corresponding to the base sequence of SEQ ID NO:33, thecomplement thereof, or an equivalent sequence containing thymine basessubstituted for uracil bases, and which preferentially hybridizes to thetarget nucleic acid or its complement under stringent hybridizationconditions. The detection probe may be capable of forming a hairpinmolecule through self-hybridization at its end portions, such as a“molecular beacon” or “molecular torch,” as described infra, under thestringent hybridization conditions. In certain embodiments the probeoptionally includes one or more detectable labels, e.g., a pair ofinteracting fluorescent labels.

For amplification reactions which do not form part of the presentinvention, the above-described promoter oligonucleotides may be modifiedto exclude the promoter sequence and/or the priming oligonucleotides maybe modified to include a promoter sequence. Additionally, where thedesired specificity for a target sequence can be achieved, the promoteroligonucleotides and/or the priming oligonucleotides described above maybe modified and used as detection probes. Also, the above-describeddetection probes may be adapted for use as amplificationoligonucleotides.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict four general methods of the present invention.

FIGS. 2A-2D depict the general methods of FIGS. 1A-1D with the furtherinclusion of an extender oligonucleotide hybridized to an extensionproduct or target sequence 3′ to the blocked promoter oligonucleotide.

FIG. 3 depicts a denaturing agarose gel showing the effect of using apromoter oligonucleotide with a 3′-blocking moiety.

FIG. 4 shows the real-time accumulation of amplification products in aMycobacterium tuberculosis system, both in the presence (FIGS. 4A, 4Cand 4E) and in the absence (FIGS. 4B, 4D and 4F) of a terminatingoligonucleotide modified to fully contain 2′-O-methyl ribonucleotides.The input target nucleic acid for these reactions was 0 copies (FIGS. 4Aand 4B), 100 copies (FIGS. 4C and 4D) and 1000 copies (FIGS. 4E and 4F).

FIG. 5 illustrates the formation of primer-dependent side-products.

FIGS. 6A and 6B illustrate the use of caps to limit side-productformation. The cap and priming oligonucleotide are separate molecules inFIG. 6A, and in FIG. 6B they are linked to each other.

FIGS. 7A and 7B depict non-denaturing agarose gels showing the effect ofa capping oligonucleotide on side-product formation. FIG. 7A depictsreactions without added template, and FIG. 7B depicts reactions withadded template.

FIG. 8 shows the real-time accumulation of amplification products in aStaphylococcus aureus system according to a method of the presentinvention. The target nucleic acid was double-stranded DNA, which wastested at four copy levels against a negative control.

FIG. 9 shows the real-time accumulation of amplification products infive different Staphylococcus aureus systems according to methods of thepresent invention. The target nucleic acid for each system wasdouble-stranded DNA, and each was system was tested at 10,000 copies oftarget against a negative control. The first system (FIG. 9A) employedthe method of FIG. 1D, and each of the remaining systems excluded acomponent of this method. The method of FIG. 9B excluded the terminatingoligonucleotide; the method of FIG. 9C excluded the displaceroligonucleotide; the method of FIG. 9D excluded the primingoligonucleotide; and the method of FIG. 9E excluded a denaturation step.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel methods, reactionmixtures, compositions and kits are provided for the amplification ofspecific nucleic acid target sequences for use in assays for thedetection and/or quantitation of such nucleic acid target sequences orfor the production of large numbers of copies of DNA and/or RNA ofspecific target sequences for a variety of uses. In particular, theembodiments of the present invention provide for amplification ofnucleic acid target sequences with enhanced specificity and sensitivity.Amplification methods of the present invention are preferably carriedout using priming oligonucleotides that target only one sense of atarget nucleic acid, with all other oligonucleotides used in theamplification methods preferably comprising a blocking moiety at their3′-termini so that they cannot be extended by a nucleic acid polymerase.

DEFINITIONS

The following terms have the following meanings unless expressly statedto the contrary. It is to be noted that the term “a” or “an” entityrefers to one or more of that entity; for example, “a nucleic acid,” isunderstood to represent one or more nucleic acids. As such, the terms“a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

1. Nucleic Acid

The term “nucleic acid” is intended to encompass a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, virus genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid. The term “polynucleotide” may beused herein to denote a nucleic acid chain. Throughout this application,nucleic acids are designated as having a 5′-terminus and a 3′-terminus.Standard nucleic acids, e.g., DNA and RNA, are typically synthesized“5′-to-3′,” i.e., by the addition of nucleotides to the 3′-terminus of agrowing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

A “non-nucleotide unit” is a unit which does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

2. Oligonucleotide

As used herein, the term “oligonucleotide” or “oligomer” is intended toencompass singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods of the present invention, as wellas subsequent detection methods. The oligonucleotide may be DNA and/orRNA and/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent, rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., it may function as a primer if it iscapable of hybridizing to a complementary strand and can further beextended in the presence of a nucleic acid polymerase, it may provide apromoter if it contains a sequence recognized by an RNA polymerase andallows for transcription, and it may function to prevent hybridizationor impede primer extension if appropriately situated and/or modified.Specific oligonucleotides of the present invention are described in moredetail below. As used herein, an oligonucleotide can be virtually anylength, limited only by its specific function in the amplificationreaction or in detecting an amplification product of the amplificationreaction. Oligonucleotides of a defined sequence and chemical structuremay be produced by techniques known to those of ordinary skill in theart, such as by chemical or biochemical synthesis, and by in vitro or invivo expression from recombinant nucleic acid molecules, e.g., bacterialor viral vectors. As intended by this disclosure, an oligonucleotidedoes not consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Modifications include basemodifications, sugar modifications or backbone modifications. Basemodifications include, but are not limited to the use of the followingbases in addition to adenine, cytidine, guanosine, thymine and uracil:C-5 propyne, 2-amino adenine, 5-methyl cytidine, inosine, and dP and dKbases. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl substitution to the ribofuranosyl moiety. SeeBecker et al., U.S. Pat. No. 6,130,038. Other sugar modificationsinclude, but are not limited to 2′-amino, 2′-fluoro,(L)-alpha-threofuranosyl, and pentopuranosyl modifications. Thenucleoside subunits may by joined by linkages such as phosphodiesterlinkages, modified linkages or by non-nucleotide moieties which do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. (DNA analogs having a pseudo peptide backbone are referred to as“peptide nucleic acids” or “PNA” and disclosed by Nielsen et al.,“Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Other linkagemodifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs (LNAs). See Imanishi etal., U.S. Pat. No. 6,268,490; and Wengel et al., U.S. Pat. No.6,670,461.) Any nucleic acid analog is contemplated by the presentinvention provided the modified oligonucleotide can perform its intendedfunction, e.g., hybridize to a target nucleic acid under stringenthybridization conditions or amplification conditions, or interact with aDNA or RNA polymerase, thereby initiating extension or transcription. Inthe case of detection probes, the modified oligonucleotides must also becapable of preferentially hybridizing to the target nucleic acid understringent hybridization conditions.

While design and sequence of oligonucleotides for the present inventiondepend on their function as described below, several variables mustgenerally be taken into account. Among the most critical are: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well known aspect ofoligonucleotide design, and various computer programs are readilyavailable to screen large numbers of potential oligonucleotides foroptimal ones.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide cannot be extended by the addition ofnucleotides to its 3′-terminus, by a DNA- or RNA-dependent DNApolymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “priming oligonucleotide.”

As used in this disclosure, the phrase “an oligonucleotide having anucleic acid sequence ‘comprising,’ ‘consisting of,’ or ‘consistingessentially of’ a sequence selected from” a group of specific sequencesmeans that the oligonucleotide, as a basic and novel characteristic, iscapable of stably hybridizing to a nucleic acid having the exactcomplement of one of the listed nucleic acid sequences of the groupunder stringent hybridization conditions. An exact complement includesthe corresponding DNA or RNA sequence.

The phrase “an oligonucleotide substantially corresponding to a nucleicacid sequence” means that the referred to oligonucleotide issufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.

One skilled in the art will understand that “substantiallycorresponding” oligonucleotides of the invention can vary from thereferred to sequence and still hybridize to the same target nucleic acidsequence. This variation from the nucleic acid may be stated in terms ofa percentage of identical bases within the sequence or the percentage ofperfectly complementary bases between the probe or primer and its targetsequence. Thus, an oligonucleotide of the present inventionsubstantially corresponds to a reference nucleic acid sequence if thesepercentages of base identity or complementarity are from 100% to about80%. In preferred embodiments, the percentage is from 100% to about 85%.In more preferred embodiments, this percentage can be from 100% to about90%; in other preferred embodiments, this percentage is from 100% toabout 95%. One skilled in the art will understand the variousmodifications to the hybridization conditions that might be required atvarious percentages of complementarity to allow hybridization to aspecific target sequence without causing an unacceptable level ofnon-specific hybridization.

3. Blocking Moiety

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be extended by a nucleic acid polymerase. A blocking moiety maybe a small molecule, e.g., a phosphate or ammonium group, or it may be amodified nucleotide, e.g., a 3′2′dideoxynucleotide or 3′deoxyadenosine5′-triphosphate (cordycepin), or other modified nucleotide. Additionalblocking moieties include, for example, the use of a nucleotide or ashort nucleotide sequence having a 3′-to-5′ orientation, so that thereis no free hydroxyl group at the 3′-terminus, the use of a 3′alkylgroup, a 3′ non-nucleotide moiety (see, e.g., Arnold et al.,“Non-Nucleotide Linking Reagents for Nucleotide Probes,” U.S. Pat. No.6,031,091, the contents of which are hereby incorporated by referenceherein), phosphorothioate, alkane-diol residues, peptide nucleic acid(PNA), nucleotide residues lacking a 3′hydroxyl group at the3′-terminus, or a nucleic acid binding protein. Preferably, the3′-blocking moiety comprises a nucleotide or a nucleotide sequencehaving a 3′-to-5′ orientation or a 3′ non-nucleotide moiety, and not a3′2′-dideoxynucleotide or a 3′ terminus having a free hydroxyl group.Additional methods to prepare 3′-blocking oligonucleotides are wellknown to those of ordinary skill in the art.

4. Binding Molecule

As used herein, a “binding molecule” is a substance which hybridizes toor otherwise binds to a target nucleic acid adjacent to or near the5′-end of the desired target sequence, so as to limit a DNA primerextension product to a desired length, i.e., a primer extension producthaving a generally defined 3′-end. As used herein, the phrase “defined3′-end” means that the 3′-end of a primer extension product is notwholly indeterminate, as would be the case in a primer extensionreaction which occurs in the absence of a binding molecule, but ratherthat the 3′-end of the primer extension product is generally known towithin a small range of bases. In certain embodiments, a bindingmolecule comprises a base region. The base region may be DNA, RNA, aDNA:RNA chimeric molecule, or an analog thereof Binding moleculescomprising a base region may be modified in one or more ways, asdescribed herein. Exemplary base regions include terminating anddigestion oligonucleotides, as described below. In other embodiments, abinding molecule may comprise, for example, a protein or drug capable ofbinding RNA with sufficient affinity and specificity to limit a DNAprimer extension product to a pre-determined length.

5. Terminating Oligonucleotide

In the present invention, a “terminating oligonucleotide” is anoligonucleotide comprising a base sequence that is complementary to aregion of the target nucleic acid in the vicinity of the 5′-end of thetarget sequence, so as to “terminate” primer extension of a nascentnucleic acid that includes a priming oligonucleotide, thereby providinga defined 3′-end for the nascent nucleic acid strand. A terminatingoligonucleotide is designed to hybridize to the target nucleic acid at aposition sufficient to achieve the desired 3′-end for the nascentnucleic acid strand. The positioning of the terminating oligonucleotideis flexible depending upon its design. A terminating oligonucleotide maybe modified or unmodified. In certain embodiments, terminatingoligonucleotides are synthesized with at least one or more 2′-O-methylribonucleotides. These modified nucleotides have demonstrated higherthermal stability of complementary duplexes. The 2′-O-methylribonucleotides also function to increase the resistance ofoligonucleotides to exonucleases, thereby increasing the half-life ofthe modified oligonucleotides. See, e.g., Majlessi et al. (1988) NucleicAcids Res. 26, 2224-9, the contents of which are hereby incorporated byreference herein. Other modifications as described elsewhere herein maybe utilized in addition to or in place of 2′-O-methyl ribonucleotides.For example, a terminating oligonucleotide may comprise PNA or an LNA.See, e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53, thecontents of which are hereby incorporated by reference herein. Althoughnot required, a terminating oligonucleotide of the present inventionpreferably includes a blocking moiety at its 3′-terminus to preventextension. A terminating oligonucleotide may also comprise a protein orpeptide joined to the oligonucleotide so as to terminate furtherextension of a nascent nucleic acid chain by a polymerase. A terminatingoligonucleotide of the present invention is typically at least 10 basesin length, and may extend up to 15, 20, 25, 30, 35, 40, 50 or morenucleotides in length. Suitable and preferred terminatingoligonucleotides are described herein. It should be noted that while aterminating oligonucleotide typically or necessarily includes a3′-blocking moiety, “3′-blocked” oligonucleotides are not necessarilyterminating oligonucleotides. Other oligonucleotides of the presentinvention, e.g., promoter oligonucleotides and capping oligonucleotidesare typically or necessarily 3′-blocked as well.

6. Modifying Oligonucleotide/Digestion Oligonucleotide

A modifying oligonucleotide provides a mechanism by which the3′-terminus of the primer extension product is determined. A modifyingoligonucleotide typically comprises a motif which hybridizes to one ormore bases in the vicinity of the 5′-end of a target sequence, and whichfacilitates termination of primer extension by means of a modifyingenzyme, e.g., a nuclease. Alternatively, a modifying oligonucleotidemight comprise a base region which hybridizes in the vicinity of the3′-end of a target sequence, and is tethered to a specific modifyingenzyme or to a chemical which can then terminate primer extension.

One specific modifying oligonucleotide is a digestion oligonucleotide. Adigestion oligonucleotide is comprised of DNA, preferably a stretch ofat least about 6 deoxyribonucleotides. The digestion oligonucleotidehybridizes to the RNA template, and the RNA of a RNA:DNA hybrid isdigested by a selective RNAse as described herein, e.g., by an enzymehaving an RNAse H activity.

7. Promoter Oligonucleotide/Promoter Sequence

As is well known in the art, a “promoter” is a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site. For binding, it was generallythought that such transcriptases required DNA which had been rendereddouble-stranded in the region comprising the promoter sequence via anextension reaction, however, the present inventors have determined thatefficient transcription of RNA can take place even under conditionswhere a double-stranded promoter is not formed through an extensionreaction with the template nucleic acid. The template nucleic acid (thesequence to be transcribed) need not be double-stranded. IndividualDNA-dependent RNA polymerases recognize a variety of different promotersequences which can vary markedly in their efficiency in promotingtranscription. When an RNA polymerase binds to a promoter sequence toinitiate transcription, that promoter sequence is not part of thesequence transcribed. Thus, the RNA transcripts produced thereby willnot include that sequence.

According to the present invention, a “promoter oligonucleotide” refersto an oligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter oligonucleotide of thepresent invention comprises a base sequence which hybridizes to a DNAtemplate, where the hybridizing sequence is situated 3′, but notnecessarily adjacent to, a promoter region. The hybridizing portion of apromoter oligonucleotide of the present invention is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. The “second region” comprises apromoter for an RNA polymerase. A promoter oligonucleotide of thepresent invention is engineered so that it is incapable of beingextended by an RNA- or DNA-dependent DNA polymerase, e.g., reversetranscriptase, preferably comprising a blocking moiety at its3′-terminus as described above. Suitable and preferred promoteroligonucleotides are described herein.

Promoter oligonucleotides of the present invention may be provided to areaction mixture with an oligodeoxynucleotide bound to aribonucleotide-containing section of the first region. Theribonucleotide-containing section preferably comprises at least 6contiguous ribonucleotides positioned at or near the 3′-end of the firstregion, and the oligodeoxynucleotide is preferably the same length asand fully complementary to the ribonucleotide-containing section of thefirst region. Upon exposure to an enzyme capable of cleaving the RNA ofan RNA:DNA duplex (e.g., an RNAse H activity), a blocking moiety at the3′-end of the promoter oligonucleotide is released and the remainder ofthe first region is in a single-stranded form which is available forhybridization to a DNA template. The remaining, uncleaved portion of thefirst region is preferably 10 to 50 nucleotides in length, as describedabove.

Promoter oligonucleotides of the present invention may be used inmethods disclosed herein to distinguish between regions of variabilityin the target sequence or between the target sequence and non-targetnucleic acid which may be present in a reaction mixture (e.g., singlenucleotide polymorphisms (“SNPs”) or mismatches between closely relatedorganisms or strains). When a primer extension product or nucleic acidsharing a high degree of sequence identity with a target nucleic acidcontains one or more base mismatches with the target binding portion ofa promoter oligonucleotide, the complements of these mismatched basesare integrated into the transcription products and the mismatched basesare then reproduced in initial and/or subsequent rounds of primerextension, thereby affecting the efficiency and sensitivity of theamplification reaction. Additionally, mismatches between the targetbinding portion of the promoter oligonucleotide and the primer extensionproduct may interfere with the formation of a double-stranded promotersequence in the presence of a reverse transcriptase, thereby furtheraffecting the efficiency of the amplification reaction. Thesedeleterious effects on an amplification reaction can be exploited todistinguish between nucleic acids exhibiting sequence variability in theregion targeted by the promoter oligonucleotide.

8. Insertion Sequence

As used herein, an “insertion sequence” is a sequence positioned betweenthe first region (i.e., template binding portion) and the second regionof a promoter oligonucleotide. Insertion sequences are preferably 5 to20 nucleotides in length, more preferably 6 to 18 nucleotides in length,and most preferably 6 to 12 nucleotides in length. The inclusion ofinsertion sequences in promoter oligonucleotides increases the rate atwhich RNA amplification products are formed. Exemplary insertionsequences are described herein.

9. Extender Oligonucleotide

An extender oligonucleotide is an oligonucleotide which hybridizes to aDNA template adjacent to or near the 3′-end of the first region of apromoter oligonucleotide. An extender oligonucleotide preferablyhybridizes to a DNA template such that the 5′-terminal base of theextender oligonucleotide is within 3, 2 or 1 bases of the 3′-terminalbase of a promoter oligonucleotide. Most preferably, the 5′-terminalbase of an extender oligonucleotide is adjacent to the 3′-terminal baseof a promoter oligonucleotide when the extender oligonucleotide and thepromoter oligonucleotide are hybridized to a DNA template. To preventextension of an extender oligonucleotide, a 3′-terminal blocking moietyis typically included. An extender oligonucleotide is preferably 10 to50 nucleotides in length, more preferably 20 to 40 nucleotides inlength, and most preferably 30 to 35 nucleotides in length.

10. Priming Oligonucleotide

A priming oligonucleotide is an oligonucleotide, at least the 3′-end ofwhich is complementary to a nucleic acid template, and which hybridizesto the template to give a priming oligonucleotide:template hybridsuitable for initiation of synthesis by an RNA- or DNA-dependent DNApolymerase. A priming oligonucleotide is extended by the addition ofcovalently bonded nucleotides to its 3′-terminus, which nucleotides arecomplementary to the template. The result is a primer extension product.A priming oligonucleotide of the present invention is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. Suitable and preferred primingoligonucleotides are described herein. Virtually all DNA polymerases(including reverse transcriptases) that are known require hybridizationof an oligonucleotide to a single-stranded template (“priming”) toinitiate DNA synthesis, whereas RNA replication and transcription(copying of RNA from DNA) generally do not require a primer. Because ofits function, a priming oligonucleotide cannot comprise a 3′-blockingmoiety that prevents extension in the presence of a DNA polymerase.Priming oligonucleotides are preferably designed to preferentiallyhybridize to a target nucleic acid, and so that it cannot be cleaved bya ribonuclease when hybridized to the target nucleic acid.

11. Displacer Oligonucleotide

A “displacer oligonucleotide” is a priming oligonucleotide whichhybridizes to a template nucleic acid upstream from a neighboringpriming oligonucleotide hybridized to the 3′-end of a target sequence(referred to herein as the “forward priming oligonucleotide”). By“upstream” is meant that a 3′-end of the displacer oligonucleotidecomplexes with the template nucleic acid 5′ to a 3′-end of the forwardpriming oligonucleotide. When hybridized to the template nucleic acid,the 3′-terminal base of the displacer oligonucleotide is preferablyadjacent to or spaced from the 5-terminal base of the forward primingoligonucleotide. More preferably, the 3′-terminal base of the displaceroligonucleotide is spaced from 5 to 35 bases from the 5′-terminal baseof the forward priming oligonucleotide. The displacer oligonucleotidemay be provided to a reaction mixture contemporaneously with the forwardpriming oligonucleotide or after the forward priming oligonucleotide hashad sufficient time to hybridize to the template nucleic acid. Extensionof the forward priming oligonucleotide can be initiated prior to orafter the displacer oligonucleotide is provided to a reaction mixture.Under amplification conditions, the displacer oligonucteotide isextended in a template-dependent manner, thereby displacing a primerextension product comprising the forward priming oligonucleotide whichis complexed with the template nucleic acid. Once displaced from thetemplate nucleic acid, the primer extension product comprising theforward priming oligonucleotide is available for complexing with apromoter oligonucleotide. The forward priming oligonucleotide and thedisplacer oligonucleotide both preferentially hybridize to the targetnucleic acid.

12. Cap or Capping Oligonucleotide

As used herein, a “cap” comprises an oligonucteotide complementary tothe 3′-end of a priming oligonucleotide, where the 5′-terminal base ofthe cap hybridizes to the 3′-terminal base of the primingoligonucleotide. A cap according to present invention is designed topreferentially hybridize to the 3′-end of the priming oligonucleotide,e.g., not with a promoter oligonucleotide, but such that the cap will bedisplaced by hybridization of the priming oligonucleotide to the targetnucleic acid. A cap may take the form of a discrete cappingoligonucleotide or it may be joined to the 5′-end of the primingoligonucleotide via a linker region, thereby forming a stem-loopstructure with the priming oligonucleotide under amplificationconditions. Such a linker region can comprise conventional nucleotides,a basic nucleotides or otherwise modified nucleotides, or anon-nucleotide region. As described in more detail herein, a suitablecap is at least three bases in length, and is no longer than about 14bases in length. Typical caps are about 5 to 7 bases in length.

13. Probe

By “probe” or “detection probe” is meant a molecule comprising anoligonucleotide having a base sequence partly or completelycomplementary to a region of an amplification product containing eithersense of a target sequence sought to be detected, so as to hybridizethereto under stringent hybridization conditions. As would be understoodby someone having ordinary skill in the art, a probe comprises anisolated nucleic acid molecule, or an analog thereof, in a form notfound in nature without human intervention (e.g., recombined withforeign nucleic acid, isolated, or purified to some extent).

The probes of this invention may have additional nucleotides outside ofthe targeted region so long as such nucleotides do not substantiallyaffect hybridization under stringent hybridization conditions and, inthe case of detection probes, do not prevent preferential hybridizationto the target sequence. A non-complementary sequence may also beincluded, such as a target capture sequence (generally a homopolymertract, such as a poly-A, poly-T or poly-U tail), promoter sequence, abinding site for RNA transcription, a restriction endonucleaserecognition site, or may contain sequences which will confer a desiredsecondary or tertiary structure, such as a catalytic active site or ahairpin structure on the probe, on the target nucleic acid, or both.

The probes preferably include at least one detectable label. The labelmay be any suitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester. Probes may also include interacting labels which emitdifferent signals, depending on whether the probes have hybridized totarget sequences. Examples of interacting labels includeenzyme/substrates, enzyme/cofactor, luminescent/quencher,luminescent/adduct, dye dimers, and Förrester energy transfer pairs.Certain probes of the present invention do not include a label. Forexample, non-labeled “capture” probes may be used to enrich for targetsequences or replicates thereof, which may then be detected by a second“detection” probe. See, e.g., Weisburg et al., “Two-Step Hybridizationand Capture of a Polynucleotide,” U.S. Pat. No. 6,534,273, the contentsof which are hereby incorporated by reference herein. While detectionprobes are typically labeled, certain detection technologies do notrequire that the probe be labeled for detection of the amplificationproduct. See, e.g., Nygren et al., “Devices and Methods for OpticalDetection of Nucleic Acid Hybridization, U.S. Pat. No. 6,060,237.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5° C. below the melting temperature of the nucleicacid duplex, and even more preferably at least 10° C. below the meltingtemperature of the nucleic acid duplex.

By “preferentially hybridize” is meant that under stringenthybridization conditions, probes of the present invention hybridize toan amplification product containing either sense of the target sequenceto form stable probe:target hybrids, while at the same time formation ofstable probe:non-target hybrids is minimized. Thus, a probe hybridizesto a target sequence or replicate thereof to a sufficiently greaterextent than to a non-target sequence, to enable one having ordinaryskill in the art to accurately quantitate the RNA replicates orcomplementary DNA (cDNA) of the target sequence formed during theamplification.

Probes of a defined sequence may be produced by techniques known tothose of ordinary skill in the art, such as by chemical synthesis, andby in vitro or in vivo expression from recombinant nucleic acidmolecules. Preferably probes are 10 to 100 nucleotides in length, morepreferably 12 to 50 bases in length, and even more preferably 18 to 35bases in length.

14. Hybridize/Hybridization

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between baseswhich are not members of these “canonical” pairs. Non-canonical basepairing is well-known in the art. (See, e.g., ROGER L. P. ADAMS ET AL.,THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (11^(th) ed. 1992).)

“Stringent hybridization conditions” or “stringent conditions” refer toconditions wherein a specific detection probe is able to hybridize to anamplification product containing either sense of a target sequence overother nucleic acids present in a reaction mixture. It will beappreciated that these conditions may vary depending upon factorsincluding the GC content and length of the probe, the hybridizationtemperature, the composition of the hybridization reagent or solution,and the degree of hybridization specificity sought. Specific stringenthybridization conditions are provided in the disclosure below.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in astable, double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. When a contiguous sequence ofnucleotides of one single-stranded region is able to form a series of“canonical” hydrogen-bonded base pairs with an analogous sequence ofnucleotides of the other single-stranded region, such that A is pairedwith U or T and C is paired with G, the nucleotides sequences are“perfectly” complementary.

By “preferentially hybridize” is meant that under stringenthybridization conditions, certain complementary nucleotide sequenceshybridize to form a stable hybrid preferentially over other, less stableduplexes.

15. Nucleic Acid “Identity”

In certain embodiments, a nucleic acid of the present inventioncomprises a contiguous base region that is at least 80%, 90%, or 100%identical to a contiguous base region of a reference nucleic acid. Forshort nucleic acids, e.g., certain oligonucleotides of the presentinvention, the degree of identity between a base region of a “query”nucleic acid and a base region of a reference nucleic acid can bedetermined by manual alignment “Identity” is determined by comparingjust the sequence of nitrogenous bases, irrespective of the sugar andbackbone regions of the nucleic acids being compared. Thus, thequery:refercnce base sequence alignment may be DNA:DNA, RNA:RNA,DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNAand DNA base sequences can be compared by replacing uridine residues (inRNA) with thymidine residues (in DNA).

16. Target Nucleic Acid/Target Sequence

A “target nucleic acid” is a nucleic acid comprising a “target sequence”to be amplified. Target nucleic acids may be DNA or RNA as describedherein, and may be either single-stranded or double-stranded. The targetnucleic acid may include other sequences besides the target sequencewhich may not be amplified. Typical target nucleic acids include virusgenomes, bacterial genomes, fungal genomes, plant genomes, animalgenomes, rRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells,mitochondrial DNA, or chromosomal DNA.

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinicalspecimens, e.g., blood, urine, saliva, feces, semen, or spinal fluid,from criminal evidence, from environmental samples, e.g., water or soilsamples, from food, from industrial samples, from cDNA libraries, orfrom total cellular RNA. By “isolated” it is meant that a samplecontaining a target nucleic acid is taken from its natural milieu, butthe term does not connote any degree of purification. If necessary,target nucleic acids of the present invention are made available forinteraction with the various oligonucleotides of the present invention.This may include, for example, cell lys is or cell permeabilization torelease the target nucleic acid from cells, which then may be followedby one or more purification steps, such as a series of isolation andwash steps. See, e.g., Clark et al., “Method for Extracting NucleicAcids from a Wide Range of Organisms,” U.S. Pat. No. 5,786,208, thecontents of which are hereby incorporated by reference herein. This isparticularly important where the sample may contain components that caninterfere with the amplification reaction, such as, for example, hemepresent in a blood sample. See Ryder et al., “Amplification of NucleicAcids from Mononuclear Cells Using Iron Complexing and Other Agents,”U.S. Pat. No. 5,639,599, the contents of which are hereby incorporatedby reference herein. Methods to prepare target nucleic acids fromvarious sources for amplification are well known to those of ordinaryskill in the art. Target nucleic acids of the present invention may bepurified to some degree prior to the amplification reactions describedherein, but in other cases, the sample is added to the amplificationreaction without any further manipulations.

The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid which is to be amplified. The “targetsequence” includes the complexing sequences to which oligonucleotides(e.g., priming oligonucleotides and/or promoter oligonucleotides)complex during the processes of the present invention. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the “target sequence”as present in the target nucleic acid. Where the “target nucleic acid”is originally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a “unique” sequence should bechosen so as to distinguish between unrelated or closely related targetnucleic acids. As will be understood by those of ordinary skill in theart, “unique” sequences are judged from the testing environment. Atleast the sequences recognized by the detection probe (as described inmore detail elsewhere herein) should be unique in the environment beingtested, but need not be unique within the universe of all possiblesequences. Furthermore, even though the target sequence should contain a“unique” sequence for recognition by a detection probe, it is not alwaysthe case that the priming oligonucleotide and/or promoteroligonucleotide are recognizing “unique” sequences. In some embodiments,it may be desirable to choose a target sequence which is common to afamily of related organisms, for example, a sequence which is common toall HIV strains that might be in a sample. In other situations, a veryhighly specific target sequence, or a target sequence having at least ahighly specific region recognized by the detection probe, would bechosen so as to distinguish between closely related organisms, forexample, between pathogenic and non-pathogenic E. coli. A targetsequence of the present invention may be of any practical length. Aminimal target sequence includes the region which hybridizes to thepriming oligonucleotide (or the complement thereof), the region whichhybridizes to the hybridizing region of the promoter oligonucleotide (orthe complement thereof), and a region used for detection, e.g., a regionwhich hybridizes to a detection probe, described in more detailelsewhere herein. The region which hybridizes to the detection probe mayoverlap with or be contained within the region which hybridizes with thepriming oligonucleotide (or its complement) or the hybridizing region ofthe promoter oligonucleotide (or its complement), In addition to theminimal requirements, the optimal length of a target sequence depends ona number of considerations, for example, the amount of secondarystructure, or self-hybridizing regions in the sequence. Determining theoptimal length is easily accomplished by those of ordinary skill in theart using routine optimization methods. Typically, target sequences ofthe present invention range from about 100 nucleotides in length to fromabout 150 to about 250 nucleotides in length. The optimal or preferredlength may vary under different conditions, which can easily be testedby one of ordinary skill in the art according to the methods describedherein. The term “amplicon” refers to a nucleic acid molecule that isgenerated during an amplification procedure and is substantiallycomplementary or identical to a sequence contained within the targetsequence.

17. Template

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase. A template may be single-stranded,double-stranded or partially double-stranded, depending on thepolymerase. The synthesized copy is complementary to the template or toat least one strand of a double-stranded or partially double-strandedtemplate. Both RNA and DNA are typically synthesized in the 5′-to-3′direction and the two strands of a nucleic acid duplex are aligned sothat the 5′-termini of the two strands are at opposite ends of theduplex (and, by necessity, so then are the 3′-termini). While accordingto the present invention, a “target sequence” is always a “template,”templates can also include secondary primer extension products andamplification products.

18. DNA-Dependent DNA Polymerase

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA copy from a DNA template. Examples are DNA polymeraseI from E. coli, bacteriophage T7 DNA polymerase, or DNA polymerases frombacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNA polymerases ofthe present invention may be the naturally occurring enzymes isolatedfrom bacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases (described below)typically also have DNA-dependent DNA polymerase activity.

19. DNA-Dependent RNA Polymerase (Transcriptase)

A “DNA-dependent RNA polymerase” or “transcriptase” is an enzyme thatsynthesizes multiple RNA copies from a double-stranded orpartially-double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

20. RNA-Dependent DNA Polymerase (Reverse Transcriptase)

An “RNA-dependent DNA polymerase” or “reverse transcriptase” (“RT”) isan enzyme that synthesizes a complementary DNA copy from an RNAtemplate. All known reverse transcriptases also have the ability to makea complementary DNA copy from a DNA template; thus, they are both RNA-and DNA-dependent DNA polymerases. RTs may also have an RNAse Hactivity. Preferred is reverse transcriptase derived from Maloney murineleukemia virus (MMLV-RT). A primer is required to initiate synthesiswith both RNA and DNA templates.

21. Selective RNAses

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes other than RNAse H which possess the same or similar activityare also contemplated in the present invention. Selective RNAses may beendonucleases or exonucleases. Most reverse transcriptase enzymescontain an RNAse H activity in addition to their polymerase activities.However, other sources of the RNAse H are available without anassociated polymerase activity. The degradation may result in separationof RNA from a RNA:DNA complex. Alternatively, a selective RNAse maysimply cut the RNA at various locations such that portions of the RNAmelt off or permit enzymes to unwind portions of the RNA. Other enzymeswhich selectively degrade RNA target sequences or RNA products of thepresent invention will be readily apparent to those of ordinary skill inthe art.

22. Sense/Antisense Strand(s)

Discussions of nucleic acid synthesis are greatly simplified andclarified by adopting terms to name the two complementary strands of anucleic acid duplex. Traditionally, the strand encoding the sequencesused to produce proteins or structural RNAs are designated as the “sense(+)” strand and its complement the “antisense (−)” strand. It is nowknown that in many cases, both strands are functional, and theassignment of the designation “sense” to one and “antisense” to theother must then be arbitrary. Nevertheless, the terms are very usefulfor designating the sequence orientation of nucleic acids and will beemployed herein for that purpose.

23. Specificity of the System

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms of anucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(i.e., the signal-to-noise ratio), described in more detail below.

24. Sensitivity

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, i.e., the ratio of specific amplicons toside-products.

25. Amplification Conditions

By “amplification conditions” is meant conditions permitting nucleicacid amplification according to the present invention. Amplificationconditions may, in some embodiments, be less stringent than “stringenthybridization conditions” as described herein. Oligonucleotides used inthe amplification reactions of the present invention hybridize to theirintended targets under amplification conditions, but may or may nothybridize under stringent hybridization conditions. On the other hand,detection probes of the present invention hybridize under stringenthybridization conditions. While the Examples section infra providespreferred amplification conditions for amplifying target nucleic acidsequences according to the present invention, other acceptableconditions to carry out nucleic acid amplifications according to thepresent invention could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

The present invention provides an autocatalytic amplification methodwhich synthesizes large numbers of RNA copies of an RNA or DNA targetsequence with high specificity and sensitivity. An important aspect ofthe present invention is the minimal production of side-products duringthe amplification. Examples of side-products include oligonucleotidedimers and self-replicating molecules. The target nucleic acid containsthe target sequence to be amplified. The target sequence is that regionof the target nucleic acid which is defined on either end by primingoligonucleotides, promoter oligonucleotides, and, optionally, a bindingmolecule, e.g., a terminating oligonucleotide or a modifyingoligonucleotide (described in more detail below), and/or the naturaltarget nucleic acid termini, and includes both the sense and antisensestrands. Promoter oligonucleotides of the present invention are modifiedto prevent the synthesis of DNA therefrom. Preferably, the promoteroligonucleotides comprise a blocking moiety attached at their 3′-terminito prevent primer extension in the presence of a polymerase. Indeed,according to the present invention, at least about 80% of theoligonucleotides present in the amplification reaction which comprise apromoter further comprise a 3′-blocking moiety. In further embodiments,at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of theoligonucleotides provided to the amplification reaction which comprise apromoter are further modified to comprise a 3′-blocking moiety. In aspecific embodiment, any oligonucleotide used in an amplificationreaction of the present invention which comprises a promoter sequencemust further comprise a 3′-terminus blocking moiety.

One embodiment of the present invention comprises amplification of atarget nucleic acid comprising an RNA target sequence. See FIGS. 1A and1B. The target nucleic acid has indeterminate 3′- and 5′-ends relativeto the desired RNA target sequence. The target nucleic acid is treatedwith a priming oligonucleotide which has a base region sufficientlycomplementary to a 3′-region of the RNA target sequence to hybridizetherewith. See Step 1 of FIGS. 1A and 1B. Priming oligonucleotides aredesigned to hybridize to a suitable region of any desired targetsequence, according to primer design methods well known to those ofordinary skill in the art. Suitable priming oligonucleotides aredescribed in more detail herein. While a priming oligonucleotide of thepresent invention can optionally include a non-hybridizing base regionsituated 5′ to the region which hybridizes with the target sequence,according to the present invention the 5′ region of a primingoligonucleotide does not include a promoter sequence recognized by anRNA polymerase. Additionally, the 5′-end of the priming oligonucleotidemay include one or modifications which improve the binding properties(e.g., hybridization or base stacking) of the priming oligonucleotide toa target sequence or RNA amplification product, as discussed more fullyinfra, provided the modifications do not substantially interfere withthe priming function of the priming oligonucleotide or cleavage of atemplate RNA to which the priming oligonucleotide is hybridized. In thepresence of nucleoside triphosphates and buffers, salts and cofactors,the 3′-end of the priming oligonucleotide is extended by an appropriateDNA polymerase, e.g., an RNA-dependent DNA polymerase (“reversetranscriptase”) in an extension reaction using the RNA target sequenceas a template to give a DNA primer extension product which iscomplementary to the RNA template. See Steps 2 and 3 of FIGS. 1A and 1B.

The DNA primer extension product is separated (at least partially) fromthe RNA template using an enzyme which degrades the RNA template. SeeStep 4 of FIGS. 1A and 1B. Suitable enzymes, i.e., “selective RNAses,”are those which act on the RNA strand of an RNA:DNA complex, and includeenzymes which comprise an RNAse H activity. Some reverse transcriptasesinclude an RNAse H activity, including those derived from Moloney murineleukemia virus (“MMLV”) and avian myeloblastosis virus (“AMV”).According to this method, the selective RNAse may be provided as anRNAse H activity of a reverse transcriptase, or may be provided as aseparate enzyme, e.g., as an E. coli RNAse H or a T. thermophilus RNAseH. Other enzymes which selectively degrade RNA present in an RNA:DNAduplex may also be used.

In certain specific embodiments, the method of the present inventionfurther comprises treating the target nucleic acid as described above tolimit the length of the DNA primer extension product to a certaindesired length. Such length limitation is typically carried out throughuse of a “binding molecule” which hybridizes to or otherwise binds tothe RNA target nucleic acid adjacent to or near the 5′-end of thedesired target sequence. See Step 1 of FIG. 1A, In certain embodiments,a binding molecule comprises a base region. The base region may be DNA,RNA, a DNA:RNA chimeric molecule, or an analog thereof. Bindingmolecules comprising a base region may be modified in one or more ways,as described elsewhere herein. Suitable binding molecules include, butare not limited to, a binding molecule comprising a terminatingoligonucleotide or a terminating protein that binds RNA and preventsprimer extension past its binding region, or a binding moleculecomprising a modifying molecule, for example, a modifyingoligonucleotide such as a “digestion” oligonucleotide that directshydrolysis of that portion of the RNA target hybridized to the digestionoligonucleotide, or a sequence-specific nuclease that cuts the RNAtarget.

A terminating oligonucleotide of the present invention has a 5′-baseregion sufficiently complementary to the target nucleic acid at a regionadjacent to, near to, or overlapping with the 5′-end of the targetsequence, to hybridize therewith. In certain embodiments, a terminatingoligonucleotide is synthesized to include one or more modifiednucleotides. For example, certain terminating oligonucleotides of thepresent invention comprise one or more 2′-O-methyl ribonucleotides, orare synthesized entirely of 2′-O-methyl ribonucleotides. See, e.g.,Majlessi et. al. (1998) Nucleic Acids Res., 26, 2224-2229. A terminatingoligonucleotide of the present invention typically also comprises ablocking moiety at its 3′-end to prevent the terminating oligonucleotidefrom functioning as a primer for a DNA polymerase. In some embodiments,the 5′-end of a terminating oligonucleotide of the present inventionoverlaps with and is complementary to at least about 2 nucleotides ofthe 5′-end of the target sequence. Typically, the 5′-end of aterminating oligonucleotide of the present invention overlaps with andis complementary to at least 3, 4, 5, 6, 7, or 8 nucleotides of the5′-end of the target sequence, but no more than about 10 nucleotides ofthe 5′-end of the target sequence. (As used herein, the term “end”refers to a 5′- or 3′-region of an oligonucleotide, nucleic acid ornucleic acid region which includes, respectively, the 5′- or 3′-terminalbase of the oligonucleotide, nucleic acid or nucleic acid region.)Suitable terminating oligonucleotides are described in more detailherein.

To the extent that a terminating oligonucleotide has a 5′ base regionwhich overlaps with the target sequence, it may be desirable tointroduce one or more base mismatches into the 5′-end of the firstregion of a promoter oligonucleotide in order to minimize or preventhybridization of the terminating oligonucleotide to the promoteroligonucleotide, as the formation of terminatingoligonucleotide:promoter oligonucleotide hybrids may negatively affectthe rate of an amplification reaction. While one base mismatch in theregion of overlap generally should be sufficient, the exact numberneeded will depend upon factors such as the length and base compositionof the overlapping region, as well as the conditions of theamplification reaction. Despite the possible benefits of a modifiedpromoter oligonucleotide, it should be noted that mutations in the firstregion of the promoter oligonucleotide could render it a poorer templatefor amplification. Moreover, it is entirely possible that in a givenamplification system the formation of terminatingoligonucleotide:promoter oligonucleotide hybrids advantageously preventsor interferes with the formation of priming oligonucleotide:promoteroligonucleotides hybrids with a 3′-end available for primer extension.See FIG. 5 (formation of primer-dependent side-products).

A modifying oligonucleotide provides a mechanism by which the3′-terminus of the primer extension product is determined. A modifyingoligonucleotide may provide a motif comprising one or more bases in thevicinity of the 5′-end of the RNA target sequence which facilitatestermination of primer extension by means of a modifying enzyme, e.g., anuclease. Alternatively, a modifying oligonucleotide might be tetheredto a specific modifying enzyme or to a chemical which can then terminateprimer extension.

One specific modifying oligonucleotide is a digestion oligonucleotide. Adigestion oligonucleotide is comprised of DNA, preferably a stretch ofat least about 6 deoxyribonucleotides. The digestion oligonucleotidehybridizes to the RNA template and the RNA of the RNA:DNA hybrid isdigested by a selective RNAse as described herein, e.g., by an RNAse Hactivity.

The single-stranded DNA primer extension product, or “first” DNA primerextension product, which has either a defined 3′-end or an indeterminate31-end, is then treated with a promoter oligonucleotide which comprisesa first region sufficiently complementary to a 3′-region of the DNAprimer extension product to hybridize therewith, a second regioncomprising a promoter for an RNA polymerase, e.g., T7 polymerase, whichis situated 5′ to the first region, e.g., immediately 5′ to or spacedfrom the first region, and modified to prevent the promoteroligonucleotide from functioning as a primer for a DNA polymerase (e.g.,the promoter oligonucleotide includes a blocking moiety attached at its3′-terminus). See Step 5 of FIGS. 1A and 1B. Upon identifying a desiredhybridizing “first region,” suitable promoter oligonucleotides can beconstructed by one of ordinary skill in the art using only routineprocedures. Those of ordinary skill in the art will readily understandthat a promoter region has certain nucleotides which are required forrecognition by a given RNA polymerase. In addition, certain nucleotidevariations in a promoter sequence might improve the functioning of thepromoter with a given enzyme, including the use of insertion sequences.

Insertion sequences are positioned between the first and second regionsof promoter oligonucleotides and function to increase amplificationrates. The improved amplification rates may be attributable to severalfactors. First, because an insertion sequence increases the distancebetween the 3′-end and the promoter sequence of a promoteroligonucleotide, it is less likely that a polymerase, e.g., reversetranscriptase, bound at the 3′-end of the promoter oligonucleotide willinterfere with binding of the RNA polymerase to the promoter sequence,thereby increasing the rate at which transcription can be initiated.Second, the insertion sequence selected may itself improve thetranscription rate by functioning as a better template for transcriptionthan the target sequence. Third, since the RNA polymerase will initiatetranscription at the insertion sequence, the primer extension productsynthesized by the priming oligonucleotide, using the RNA transcriptionproduct as a template, will contain the complement of the insertionsequence toward the 3′-end of the primer extension product. By providinga larger target binding region, i.e., one which includes the complementof the insertion sequence, the promoter oligonucleotide may bind to theprimer extension product faster, thereby leading to the production ofadditional RNA transcription products sooner. Insertion sequences arepreferably 5 to 20 nucleotides in length and should be designed tominimize intramolecular folding and intermolecular binding with otheroligonucleotides present in the amplification reaction mixture. Programswhich aid in minimizing secondary structure are well known in the artand include Michael Zucker's mfold software for predicting RNA and DNAsecondary structure using nearest neighbor thermodynamic rules. Thelatest version of Michael Zucker's mfold software can be accessed on theWeb at www.bioinfo.rpi.edu/applications/mfold using a hypertext transferprotocol (http) in the URL. Currently preferred insertion sequencesinclude the nucleotide sequences of SEQ ID Nos. 1 and 2 in combinationwith the T7 RNA polymerase promoter sequence of SEQ ID NO:3. See Ikedaet at. (1992) J. Biol. Chem. 267, 2640-2649. Other useful insertionsequences may be identified using in vitro selection methods well knownin the art.

Assaying promoter oligonucleotides with variations in the promotersequences is easily carried out by the skilled artisan using routinemethods. Furthermore, if it is desired to utilize a different RNApolymerase, the promoter sequence in the promoter oligonucleotide iseasily substituted by a different promoter. Substituting differentpromoter sequences is well within the understanding and capabilities ofthose of ordinary skill in the art. It is important to note thataccording to the present invention, promoter oligonucleotides providedto the amplification reaction mixture are modified to prevent theinitiation of DNA synthesis from their 3′-termini, and preferablycomprise a blocking moiety attached at their 3′-termini. Furthermore,terminating oligonucleotides and capping oligonucleotides, and evenprobes used in the methods of the present invention also optionallycomprise a blocking moiety attached at their 3′-termini.

Where a terminating oligonucleotide is used, the first region of thepromoter oligonucleotide is designed to hybridize with a desired 3′-endof the DNA primer extension product with substantial, but notnecessarily exact, precision. Subsequently, the second region of thepromoter oligonucleotide may act as a template, allowing the first DNAprimer extension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide,i.e., the region comprising the promoter sequence, rendering thepromoter double-stranded. See Steps 6 and 7 of FIG. 1A. An RNApolymerase which recognizes the promoter binds to the promoter sequence,and initiates transcription of multiple RNA copies complementary to theDNA primer extension product, which copies are substantially identicalto the target sequence. By “substantially identical” it is meant thatthe multiple RNA copies may have additional nucleotides either 5′ or 3′relative to the target sequence, or may have fewer nucleotides either 5′or 3′ relative to the target sequence, depending on, e.g., theboundaries of “the target sequence,” the transcription initiation point,or whether the priming oligonucleotide comprises additional nucleotides5′ of the primer region (e.g., a linked “cap” as described herein).Where a target sequence is DNA, the sequence of the RNA copies isdescribed herein as being “substantially identical” to the targetsequence. It is to be understood, however, that an RNA sequence whichhas uridine residues in place of the thymidine residues of the DNAtarget sequence still has a “substantially identical” sequence. The RNAtranscripts so produced may automatically recycle in the above systemwithout further manipulation. Thus, this reaction is autocatalytic. Inthose embodiments where a binding molecule or other means forterminating a primer extension reaction is not used, the first region ofthe promoter oligonucleotide is designed to hybridize with a selectedregion of the first DNA primer extension product which is expected to be5′ to the 3′-terminus of the first DNA primer extension product, butsince the 3′-terminus of the first DNA primer extension product isindeterminate, the region where the promoter oligonucleotide hybridizesprobably will not be at the actual 3′-end of the first DNA primerextension product. According to this embodiment, it is generally thecase that at least the 3′-terminal base of the first DNA primerextension product does not hybridize to the promoter oligonucleotide.See Step 5 of FIG. 1B. Thus, according to this embodiment the first DNAprimer extension product will likely not be further extended to form adouble-stranded promoter.

Surprisingly, the inventors discovered that the formation of adouble-stranded promoter sequence through extension of a templatenucleic acid is not necessary to permit initiation of transcription ofRNA complementary to the first DNA primer extension product. See Step 6of FIG. 1B. The resulting “first” ′ RNA products are substantiallyidentical to the target sequence, having a 5′-end defined by thetranscription initiation point, and a 3′-end defined by the 5′-end ofthe first DNA primer extension product. See Step 7 of FIG. 1B. Asillustrated in FIG. 1B, a sufficient number of first RNA products areproduced to automatically recycle in the system without furthermanipulation. The priming oligonucleotide hybridizes to the 3′-end ofthe first RNA products, and is extended by a DNA polymerase to form asecond DNA primer extension product. Unlike the first DNA primerextension product formed without the use of a terminatingoligonucleotide or other binding molecule, the second DNA primerextension product has a defined 3′-end which is complementary to the5′-ends of the first RNA products. See Steps 8-10 of FIG. 1B. The secondDNA primer extension product is separated (at least partially) from theRNA template single-stranded second DNA primer extension product is thentreated with a promoter oligonucleotide as described above, and thesecond region of the promoter oligonucleotide acts as a template,allowing the second DNA primer extension product to be further extendedto add a base region complementary to the second region of the promoteroligonucleotide, i.e., the region comprising the promoter sequence,rendering the promoter double-stranded. See Steps 12-14 of FIG. 1B. AnRNA polymerase which recognizes the promoter binds to the promotersequence, and initiates transcription of multiple “second” RNA productscomplementary to the second DNA primer extension product, andsubstantially identical to the target sequence. See Step 15 of FIG. 1B.The second RNA transcripts so produced automatically recycle in theabove system without further manipulation. Thus, this reaction isautocatalytic. See Steps 7-15 of FIG. 1B.

In another embodiment, the present invention is drawn to a method ofsynthesizing multiple copies of a target sequence from a target nucleicacid comprising a DNA target sequence. This embodiment is diagramed inFIG. 1C. The target nucleic acid may be either single-stranded,partially single-stranded, or double-stranded DNA. When the DNA isdouble-stranded, it is denatured, or partially denatured, prior toamplification. The DNA target nucleic acid need not have a defined3′-end. The single-stranded, partially single-stranded, or denatured DNAtarget nucleic acid is treated with a promoter oligonucleotide asdescribed above. The first region of the promoter oligonucleotide isdesigned to hybridize with a selected region of the target nucleic acidin the 3′-region of the desired target sequence, but since the 3′-end ofthe target nucleic acid need not be coterminal with the 3′-end of thetarget sequence, the region where the promoter oligonucleotidehybridizes will likely not be at or near the 3′-end of the targetnucleic acid sequence. See Step 1 of FIG. 1C. Thus, the promoter regionof the promoter oligonucleotide will likely remain single-stranded.

As noted above, the inventors surprisingly discovered that it is notnecessary for the single-stranded promoter sequence on the promoteroligonucleotide to form a double-stranded promoter through extension ofa template nucleic acid in order for the promoter sequence to berecognized by the corresponding RNA polymerase and, in this case,initiate transcription of RNA complementary to the DNA target sequence.See Step 2 of FIG. 1C. The resulting “first RNA products” have a 5′-enddefined by the transcription initiation point for the promoter, however,the 3′-region will remain indeterminate. See Step 3 of FIG. 1C. Thesefirst RNA products are then treated with a priming oligonucleotide. Thepriming oligonucleotide hybridizes to a region of the first RNA productsat a position complementary to a 5′ region of the desired targetsequence, and is extended by a DNA polymerase to form a DNA primerextension product. See Steps 4-6 of FIG. 1C. This DNA primer extensionproduct has a 5′-end coinciding with the 5′-end of the primingoligonucteotide, and a 3′-end coinciding with the 5′-end of the firstRNA products. See Step 6 of FIG. 1C. The DNA primer extension product isseparated (at least partially) from its RNA template using an enzymewhich selectively degrades the RNA template, as described above. SeeStep 7 of FIG. 1C. The DNA primer extension product is then treated withthe promoter oligonucleotide, as described above, and the second regionof the promoter oligonucleotide acts as a template, allowing the DNAprimer extension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide,i.e., the region comprising the promoter, rendering the promoterdouble-stranded. See Steps 8-10 of FIG. 1C. An RNA polymerase whichrecognizes the promoter binds to the promoter sequence, and initiatestranscription of multiple RNA pro ducts complementary to the DNA primerextension product. See Step 11 of FIG. 1C. The sequence of these “secondRNA products” is substantially complementary to the desired targetsequence. The RNA products so produced automatically recycle in theabove system without further manipulation. Thus, this reaction isautocatalytic. See Steps 3-11 of FIG. 1C.

In yet another embodiment, the present invention relates to a method ofsynthesizing multiple copies of a target sequence from a target nucleicacid comprising a DNA target sequence. One aspect of this embodiment isillustrated in FIG. 1D. Prior to amplification, the target nucleic acidin this embodiment may be single-stranded, partially single-stranded, ordouble-stranded. If a region of the target nucleic acid containing thetarget sequence is double-stranded, then the target nucleic acid may bedenatured, or partially denatured, prior to initiating amplification torender the target sequence accessible to the priming oligonucleotide.See Step 1 of FIG. 1D. Denaturation may be effected by heat, high pH,and/or low ionic strength. (Useful chemical denaturants includeimidazole, dimethyl sulfoxide, formamide, urea and/or sodiumhydryoxide.) The accessible target sequence is treated with a primingoligonucleotide, as described infra, which hybridizes to a 3′-end of thetarget sequence. See Step 2 of FIG. 1D. In the presence of nucleosidetriphosphates and buffers, salts and cofactors, the 3′-end of thepriming oligonucleotide is extended by a DNA polymerase in an extensionreaction using the DNA target sequence as a template to give a first DNAprimer extension product that is complementary to the template. SeeSteps 3 and 4 of FIG. 1D. In one aspect of this embodiment, a bindingmolecule is included which limits the length of the first DNA primerextension product by hybridizing or otherwise binding to the targetnucleic acid adjacent to or near the 5′-end of the target sequence. Thebinding molecule may be any of the binding molecules described hereinthat is appropriate for a target nucleic acid containing a DNA targetsequence, but is preferably a terminating oligonucleotide. See Steps 2to 4 of FIG. 1D. One advantage of a terminating oligonucleotide is thatit does not require the use of a restriction endonuclease to create adefined 3′-end on the first DNA primer extension product.

To separate the first DNA primer extension product from the template,the target nucleic acid can be treated with a displacer oligonucleotidein another aspect of this embodiment. See Step 5 of FIG. 1D. Thedisplacer oligonucleotide has a priming function and is designed tohybridize to the target nucleic acid upstream from the primingoligonucleotide (referred to as the “forward priming oligonucleotide” inthis embodiment), By “upstream” is meant that a 3′-end of the displaceroligonucleotide hybridizes to the target nucleic acid upstream from a3′-end of the forward priming oligonucleotide. Thus, the displaceroligonucleotide and the forward priming oligonucleotide may hybridize tooverlapping or distinct regions of the target nucleic acid. In preferredembodiments, the 3′-terminus of the displacer oligonucleotide isadjacent to or spaced up to 5 to 35 bases from the 5′-terminus of theforward priming oligonucleotide relative to the target nucleic acid(i.e., the target nucleic acid has up to 5 to 35, contiguous unboundnucleotides situated between the 3′-terminal base of the displaceroligonucleotide and the 5′-terminal base of the priming oligonucleotidewhen both oligonucleotides are hybridized to the target nucleic acid).The displacer oligonucleotide is generally from 10 to 50 nucleotides inlength and may include one or more modifications at the 5′-end whichimprove the binding properties (e.g., hybridization or base stacking) ofthe displacer oligonucleotide to the target nucleic acid, provided thatthe modifications do not substantially interfere with the primingfunction of the displacer oligonucleotide. The displacer oligonucleotideand the forward priming oligonucleotide are designed to hybridize to thetarget nucleic acid under the same conditions. The target nucleic acidis preferably treated with the displacer oligonucleotide after theforward priming oligonucleotide has had sufficient time to hybridize tothe target nucleic acid. Alternatively, the target nucleic acid istreated with both the displacer oligonucleotide and the forward primingoligonucleotide before exposing the mixture to a polymerase suitable forextending the 3′-ends of the displacer oligonucleotide and the forwardpriming oligonucleotide. In the presence of the DNA polymerase, the3′-end of the displacer oligonucleotide is extended in atemplate-dependent manner to form a second DNA primer extension productwhich displaces the first DNA primer extension product from the targetnucleic acid, thereby making it available for hybridization to apromoter oligonucleotide. See Steps 6 and 7 of FIG. 1D. In analternative approach, conditions could be established whereby thepromoter oligonucleotide gains access the first DNA primer extensionproduct through stand invasion facilitated by, for example, DNAbreathing (e.g., AT rich regions), low salt conditions, and/or the useof DMSO and/or osmolytes, such as betaine. The promoter oligonucleotideof this embodiment is the same as that described above and, likewise, ismodified to prevent the promoter oligonucleotide from functioning as apriming oligonucleotide for a DNA polymerase (e.g., the promoteroligonucteotide includes a blocking moiety at its 3′-terminus).

Where a terminating oligonucleotide or other binding molecule capable ofdetermining the 3′-end of the first DNA primer extension product isused, a first region of the promoter oligonucleotide is designed tohybridize to the 3′-end of the first DNA primer extension product withsufficient precision that a second region of the promoteroligonucleotide acts as a template, allowing the first DNA primerextension product to be further extended to add a base regioncomplementary to the second region of the promoter oligonucleotide. SeeSteps 8-10 of FIG. 1D. This extension reaction renders a regioncontaining the promoter sequence double-stranded. An RNA polymerasewhich recognizes the promoter then binds to the promoter sequence andinitiates transcription of multiple RNA copies complementary to thefirst DNA primer extension product, where the RNA copies aresubstantially identical to the target sequence, as this term is definedhereinabove. See Steps 11 and 12 of FIG. 1D. The RNA transcripts soproduced may automatically recycle in the method of this embodiment and,thus, the reaction is autocatalytic. See Steps 9-17 of FIG. 1D. In thesubsequent rounds of amplification, a third DNA primer extension productcomprising the promoter oligonucleotide and complementary to the RNAtranscript is formed, and this DNA primer extension product is thenseparated from the RNA transcript (at least partially) using an enzymewhich selectively degrades the RNA transcript (e.g., an enzyme having anRNase H activity, such as a reverse transcriptase derived from MMLV orAMV). See Steps 13-16 of FIG. 1D.

In those aspects of this embodiment where a binding molecule or othermeans for terminating a primer extension reaction are not used, thefirst region of the promoter oligonucleotide is designed to hybridize toa selected region of the first DNA primer extension product which isexpected to be 5′ to the 3′-terminus of the first DNA primer extensionproduct. Since the 3′-terminus of the first DNA primer extension productis indeterminate, the region where the promoter oligonucleotidehybridizes probably will not be at the actual 3′-end of the first DNAprimer extension product. According to this embodiment, it is generallythe case that at least the 3′-terminal base of the first DNA primerextension product does not hybridize to the promoter oligonucleotide.Thus, according to this embodiment the first DNA primer extensionproduct will likely not be further extended to form a double-strandedpromoter As discussed above, the inventors surprisingly discovered thatthe formation of a double-stranded promoter sequence through extensionof a template nucleic acid is not necessary to permit initiation oftranscription of RNA complementary to the first DNA primer extensionproduct. In subsequent rounds of amplification, however, the RNAtranscripts will include a defined 3′-end and DNA primer extensionproducts complementary to the RNA transcripts will hybridize to thepromoter oligonucleotide and be extended to add a sequence complementaryto a region of the promoter oligonucleotide containing the promotersequence.

The inventors also discovered that the rate of amplification could beenhanced by providing an extender oligonucleotide to a reaction mixture,as diagramed in FIGS. 2A-2D. (Step 5 of FIGS. 2A and 2B, Step 4 of FIG.2C, and Step 8 of FIG. 2D.) An extender oligonucleotide is generally 10to 50 nucleotides in length and hybridizes to a DNA template (i.e., theDNA target sequence or any of the DNA primer extension productsdescribed herein) downstream from a promoter oligonucleotide. Whenincluded, the 5′-terminal base of the extender oligonucleotide ispositioned near or adjacent to the 3′-terminal base of the promoteroligonucleotide when both oligonucleotides are hybridized to a DNAtemplate. (By “adjacent to” is meant that the DNA template has nounbound bases situated between the 3′-terminal base of the promoteroligonucleotide and the 5′-terminal base of the extender oligonucleotidewhen both oligonucleotides are hybridized to the DNA template.) Mostpreferably, the extender oligonucleotide hybridizes to a DNA templatesuch that the 5′-terminal base of the extender oligonucleotide is spacedno more than three nucleotides from the 3′-terminal base of the promoteroligonucleotide relative to the DNA template (i.e., the DNA template hasa maximum of three, contiguous unbound nucleotides situated between the3′-terminal base of the promoter oligonucleotide and the 5′-terminalbase of the extender oligonucleotide when both oligonucleotides arehybridized to the DNA template). To prevent the extender oligonucleotidefrom functioning as a priming oligonucleotide in a primer extensionreaction) the extender oligonucleotide preferably includes a 3′-terminalblocking moiety. While not wishing to be bound by theory, it is believedthat the phosphate at the 3′-end of the extender oligonucleotidefunctions to draw the DNA-dependent DNA polymerase (e.g., reversetranscriptase) farther away from the promoter sequence of the promoteroligonucleotide, thereby minimizing interference with the binding andprogress of the RNA polymerase in transcription. It is also possiblethat the extender oligonucleotide facilitates faster transcriptionreactions by limiting secondary structure within the target sequence.

In one aspect, the present invention relates to minimizing side-productformation in nucleic acid amplification reactions. One type ofside-product is referred to herein as an “oligonucleotide dimer.” Thisside-product occurs when a priming oligonucleotide base-pairsnon-specifically with another nucleic acid in the amplificationreaction, e.g., the promoter oligonucleotide. Since the primingoligonucleotide can be extended via a DNA polymerase, a double-strandedform of the promoter oligonucleotide can result, which can betranscribed into non-specific, amplifiable side-products. To preventpriming oligonucleotides from participating in the formation ofoligonucleotide dimers, one option is to add a short complementarynucleotide “cap” to the 3′-end of the priming oligonucleotide. See FIGS.6A and 6B. A cap is thought to reduce non-specific hybridization betweenthe priming oligonucleotide and other nucleic acids in the reaction,e.g., the promoter oligonucleotide, thereby eliminating or substantiallyreducing the production of “oligonucleotide-dimer” side-products ascompared to amplification reactions carried out under identicalconditions, but without the use of a cap. As used herein, a capcomprises a base region complementary to a region at the 3′-end of thepriming oligonucleotide which is preferably pre-hybridized to thepriming oligonucleotide prior to its introduction into an amplificationreaction mixture. A suitable cap length will vary based on base content,stringency conditions, etc., but will typically hybridize to up to 3, 6,9, 12, 15, 18, or 20 contiguous or non-contiguous nucleotides at the3′-end of the priming oligonucleotide. Suitable caps preferably rangefrom 5 to 10 bases in length. The length of the complementary cap regionis dependent on several variables, for example, the melting temperatureof the double-stranded hybrid formed with the 3′-end of the primingoligonucleotide. In general, an efficient cap will specificallyhybridize to a region at the 3′-end of the priming oligonucleotide morestrongly than any non-specific reactions with other oligonucleotidespresent in the amplification reaction, but will be readily displaced infavor of specific hybridization of the priming oligonucleotide with thedesired template. Exemplary caps comprise, or alternately consistessentially of, or alternately consist of an oligonucleotide from 5 to 7bases in length which hybridizes to a region at the 3′-end of thepriming oligonucleotide, such that the 5′-terminal base of the caphybridizes to the 3′-terminal base of the priming oligonucleotide.Typically, a cap will hybridize to no more than 8, 9, or 10 nucleotidesof a region at the 3′-end of the priming oligonucleotide.

A cap may take the form of a capping oligonucleotide or a base regionattached to the 5′-end of the priming oligonucleotide, either directlyor through a linker. See FIGS. 6A and 6B. A capping oligonucleotide issynthesized as a separate oligonucleotide from the primingoligonucleotide, and normally comprises a blocking moiety at its3′-terminus to prevent primer extension by a DNA polymerase, asillustrated in FIG. 6A. Alternatively, the cap comprises a base regioncomplementary to a region at the 3′-end of the priming oligonucleotide,which is connected to the 5′-end of the priming oligonucleotide via alinking region comprising, alternately consisting essentially of, oralternately consisting of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. SeeFIG. 6B. Typically, the nucleotides in the linking region are a basicnucleotides. By “a basic nucleotide” is meant a nucleotide comprising aphosphate group and a sugar group, but not a base group. Constructing apriming oligonucleotide with a cap attached to its 5′-end simplifiesoligonucleotide synthesis by requiring the synthesis of only a singleoligonucleotide comprising both the priming portion and the cap.

The caps described herein may also be used with other oligonucleotidesprovided to a reaction mixture, including displacer and extenderoligonucleotides, which have free 3′-hydroxyls that are capable ofparticipating in an extension reaction in the presence of a DNApolymerase. The importance of caps increases with the number ofoligonucleotides having free 3′-hydroxyls.

In any of the embodiments described above, once a desired region for thetarget sequence is identified, that region can be analyzed to determinewhere selective RNAse degradation will optimally cause cuts or removalof sections of RNA from the RNA:DNA duplex. Analyses can be conducted todetermine the effect of the RNAse degradation of the target sequence byRNAse H activity present in AMV reverse transcriptase or MMLV reversetranscriptase, by an exogenously added selective enzyme with an RNAseactivity, e.g., E. coli RNAse H, or selective enzymes with an RNAseactivity from other sources, and by combinations thereof. Following suchanalyses, the priming oligonucleotide can be selected for so that itwill hybridize to a section of RNA which is substantially nondegraded bythe selective RNAse present in the reaction mixture, because substantialdegradation at the binding site for the priming oligonucleotide couldinhibit initiation of DNA synthesis and prevent optimal extension of theprimer. In other words, a priming oligonucleotide is typically selectedto hybridize to a region of an RNA target nucleic acid or the complementof a DNA target nucleic acid, located so that when the RNA is subjectedto selective RNAse degradation, there is no substantial degradationwhich would prevent formation of the primer extension product.

Conversely, the site for hybridization of the promoter oligonucleotidemay be chosen so that sufficient degradation of the RNA strand occurs topermit efficient hybridization of the promoter oligonucleotide to theDNA strand. Typically, only portions of RNA are removed from the RNA:DNAduplex through selective RNAse degradation and, thus, some parts of theRNA strand will remain in the duplex. Selective RNAse degradation on theRNA strand of an RNA:DNA hybrid results in the dissociation of smallpieces of RNA from the hybrid. Positions at which RNA is selectivelydegraded may be determined through standard hybridization analyses.Thus, a promoter oligonucleotide may be selected which will moreefficiently bind to the DNA after selective RNAse degradation, i.e.,will bind at areas where RNA fragments are selectively removed.

FIGS. 1A-1D and 2A-2D do not show the RNA portions which may remainafter selective RNAse degradation. It is to be understood, however, thateven though FIGS. 1A-1D and 2A-2D show complete removal of RNA from theDNA:RNA duplex, under certain conditions only partial removal actuallyoccurs. Indeed, amplification as depicted in FIGS. 1A-1D and 2A-2D maybe inhibited if a substantial portion of the RNA strand of an RNA:DNAhybrid remains undegraded, thus preventing hybridization of the promoteroligonucleotide and/or the optional extender oligonucleotide. However,based upon principles and methods disclosed in this application, as wellas those disclosed by Kacian et al, U.S. Pat. No. 5,339,491, routinemodifications can be made by those skilled in the art according to theteachings of this invention to provide an effective and efficientprocedure for amplification of RNA.

In summary, the present invention provides methods for autocatalyticallysynthesizing multiple copies of a target sequence from a target nucleicacid without repetitive manipulation of reaction conditions such astemperature, ionic strength and pH, which comprises combining into areaction mixture a target nucleic acid which comprises either an RNAtarget sequence, or a single-stranded or partially single-stranded DNAtarget sequence or a double-stranded DNA sequence which has beenrendered at least partially single-stranded; a priming oligonucleotide,a promoter oligonucleotide, and, optionally, a displaceroligonucleotide, an extender oligonucleotide and/or a binding moleculeor other means for terminating a primer extension reaction, all asdescribed above; a reverse transcriptase or an RNA-dependent DNApolymerase and a DNA-dependent DNA polymerase; an enzyme activity whichselectively degrades the RNA strand of an RNA:DNA complex (such as anRNAse H activity); and an RNA polymerase which recognizes the promotersequence in the promoter oligonucleotide. The reaction mixture alsoincludes the necessary building blocks for nucleic acid amplification,e.g., nucleoside triphosphates, buffers, salts, and stabilizing agents.The components of the reaction mixture may be combined stepwise or atonce. The reaction mixture is incubated under conditions whereby anoligonucleotide:target nucleic acid is formed, and DNA priming andnucleic acid synthesis can occur for a period of time sufficient toallow multiple copies of the target sequence or its complement to beproduced. The reaction advantageously takes place under conditionssuitable for maintaining the stability of reaction components, such asthe enzymes, and without requiring modification or manipulation ofreaction conditions during the course of the amplification reaction.Accordingly, the reaction of some embodiments may take place underconditions that are substantially isothermal and include substantiallyconstant ionic strength and pH.

As such, the amplification methods of the present invention do notrequire repeated denaturation steps to separate the RNA:DNA complexesproduced upon extension of the priming oligonucleotide. A denaturationstep would require manipulation of reaction conditions, such as bysubstantially increasing the temperature of the reaction mixture(generally from ambient temperature to a temperature between about 80°C. and about 105° C.), reducing its ionic strength (generally by 10× ormore) or changing pH (usually increasing pH to 10 or greater). Suchmanipulations of the reaction conditions often deleteriously affectenzyme activities, requiring addition of additional enzyme and alsonecessitate further manipulations of the reaction mixture to return itto conditions suitable for further nucleic acid synthesis. In thoseembodiments where the target nucleic acid is double-stranded DNA, aninitial denaturation step is often required. Denaturation may be carriedout by altering temperature, ionic strength, and/or pH as describedabove, prior to adding the remaining components of the reaction mixture.Once the remaining components are added, no additional manipulations ofthe reaction mixture are needed.

The methods of the present invention are designed to decrease, diminish,or substantially eliminate side-product formation in the amplificationreactions. For example, side-products are decreased, diminished, orsubstantially eliminated through the utilization of promoteroligonucleotides modified to prevent primer extension by a DNApolymerase, generally by including a blocking moiety at the 3′-terminiof the promoter oligonucleotides. Further embodiments decrease,diminish, or substantially eliminate side-products through the use of acap which hybridizes to a region at the 3′-end of the primingoligonucleotide, thereby preventing oligonucleotide dimer formation.According to the present invention, most, e.g., at least about 90%, ofthe oligonucleotides present in the amplification reaction whichcomprise a promoter further comprise a 3′-blocking moiety to preventprimer extension. In a specific embodiment, any oligonucleotide used inthe amplification reaction which comprises a promoter, not just thepromoter oligonucleotide, further comprises a 3′-blocking moiety. Incertain preferred embodiments, most, e.g., at least about 80%, 90%, 95%,96%, 97%, 98% or 99%, or all oligonucleotides required for theamplification reaction, other than the priming oligonucleotides(including displacer oligonucleotides, if used), comprise a 3′-blockingmoiety. Thus, in certain embodiments, most if not all DNA polymeraseactivity in the amplification reactions is limited to the formation ofDNA primer extension products which comprise the primingoligonucleotides.

Promoters or promoter sequences suitable for incorporation in promoteroligonucleotides used in the methods of the present invention arenucleic acid sequences (either naturally occurring, producedsynthetically or a product of a restriction digest) that arespecifically recognized by an RNA polymerase that recognizes and bindsto that sequence and initiates the process of transcription, whereby RNAtranscripts are produced. Typical, known and useful promoters includethose which are recognized by certain bacteriophage polymerases, such asthose from bacteriophage T3, 17, and SP6, and a promoter from E. coli.The sequence may optionally include nucleotide bases extending beyondthe actual recognition site for the RNA polymerase which may impartadded stability or susceptibility to degradation processes or increasedtranscription efficiency. Promoter sequences for which there is a knownand available polymerase that is capable of recognizing the initiationsequence are particularly suitable to be employed.

Suitable DNA polymerases include reverse transcriptases. Particularlysuitable DNA polymerases include AMV reverse transcriptase and MMLVreverse transcriptase. Some of the reverse transcriptases suitable foruse in the methods of the present invention, such as AMV and MMLVreverse transcriptases, have an RNAse H activity. Indeed, according tocertain embodiments of the present invention, the only selective RNAseactivity in the amplification reaction is provided by the reversetranscriptase—no additional selective RNAse is added. However, in somesituations it may also be useful to add an exogenous selective RNAse,such as E. coli RNAse H. Although the addition of an exogenous selectiveRNAse is not required, under certain conditions, the RNAse H activitypresent in, e.g., AMV reverse transcriptase may be inhibited orinactivated by other components present in the reaction mixture. In suchsituations, addition of an exogenous selective RNAse may be desirable.For example, where relatively large amounts of heterologous DNA arepresent in the reaction mixture, the native RNAse H activity of the AMVreverse transcriptase may be somewhat inhibited and thus the number ofcopies of the target sequence produced accordingly reduced. Insituations where the target nucleic acid comprises only a small portionof the nucleic acid present (e.g., where the sample contains significantamounts of heterologous DNA and/or RNA), it is particularly useful toadd an exogenous selective RNAse. See, e.g., Kacian et al, U.S. Pat. No.5,399,491, the contents of which are hereby incorporated by referenceherein (see Example 8).

RNA amplification products produced by the methods described above mayserve as templates to produce additional amplification products relatedto the target sequence through the above-described mechanisms. Thesystem is autocatalytic and amplification by the methods of the presentinvention occurs without the need for repeatedly modifying or changingreaction conditions such as temperature, pH, ionic strength and thelike. These methods do not require an expensive thermal cyclingapparatus, nor do they require several additions of enzymes or otherreagents during the course of an amplification reaction.

The methods of the present invention are useful in assays for detectingand/or quantitating specific nucleic acid target sequences in clinicalenvironmental, forensic, and similar samples or to produce large numbersof RNA amplification products from a specific target sequence for avariety of uses. For example, the present invention is useful to screenclinical samples (e.g., blood, urine, feces, saliva, semen, or spinalfluid), food, water, laboratory and/or industrial samples for thepresence of specific nucleic acids. The present invention can be used todetect the presence of, for example, viruses, bacteria, fungi, orparasites. The present invention is also useful for the detection ofhuman, animal, or plant nucleic acids for genetic screening, or incriminal investigations, archeological or sociological studies.

In a typical assay, a sample containing a target nucleic acid to beamplified is mixed with a buffer concentrate containing the buffer,salts, magnesium, triphosphates, oligonucleotides, e.g., a primingoligonucleotide, a promoter oligonucleotide, and, optionally, adisplacer oligonucleotide, an extender oligonucleotide and/or a bindingmolecule, e.g., a terminating oligonucleotide or a digestionoligonucleotide, and/or a capping oligonucleotide, and other reagents.The reaction may optionally be incubated at a temperature, e.g., 60-100°C., for a period of time sufficient to denature any secondary structuresin the target nucleic acid or to denature a double-stranded DNA targetnucleic acid. After cooling, reverse transcriptase, an RNA polymerase,and, if desired, a separate selective RNAse, e.g., RNAse H, are addedand the reaction is incubated for a specified amount of time, e.g., fromabout 10 minutes to about 120 minutes, at an optimal temperature, e.g.,from about 20° C. to about 55° C., or more, depending on the reagentsand other reaction conditions. The displacer oligonucleotide, if used,may be provided with the enzymes to permit sufficient time for thepriming oligonucleotide to hybridize to the target nucleic acid beforeinitiating amplification.

The amplification product can be detected by hybridization with anoptionally labeled detection probe and measurement of the resultinghybrids can be performed in any conventional manner. Design criteria inselecting probes for detecting particular target sequences are wellknown in the art and are described in, for example, Hogan et al.,“Methods for Making Oligonucleotide Probes for the Detection and/orQuantitation of Non-Viral Organisms,” U.S. Pat. No. 6,150,517, thecontents of which are hereby incorporated by reference herein. Hoganteaches that probes should be designed to maximize homology for thetarget sequence(s) and minimize homology for possible non-targetsequences. To minimize stability with non-target sequences. Hoganinstructs that guanine and cytosine rich regions should be avoided, thatthe probe should span as many destabilizing mismatches as possible, andthat the length of perfect complementarity to a non-target sequenceshould be minimized. Contrariwise, stability of the probe with thetarget sequence(s) should be maximized, adenine and thymine rich regionsshould be avoided, probe:target hybrids are preferably terminated withguanine and cytosine base pairs, extensive self-complementarity isgenerally to be avoided, and the melting temperature of probe:targethybrids should be about 2-10° C. higher than the assay temperature.

In particular, the amplification product can be assayed by theHybridization Protection Assay (“HPA”), which involves hybridizing achemiluminescent oligonucleotide probe to the target sequence, e.g., anacridinium ester-labeled (“AE”) probe, selectively hydrolyzing thechemiluminescent label present on unhybridized probe, and measuring thechemiluminescence produced from the remaining probe in a luminometer.See, e.g., Arnold et al., “Homogenous Protection Assay,” U.S. Pat. No.5,283,174 and NORMAN C. NELSON ET AL., NONISOTOPIC PROBING, BLOTTING,AND SEQUENCING, ch. 17 (Larry J. Kricka ed., 2d ed. 1995), each of whichis hereby incorporated by reference in its entirety. Particular methodsof carrying out HPA using AE probes are disclosed in the Examplessection hereinbelow.

In further embodiments, the present invention provides quantitativeevaluation of the amplification process in realtime by methods describedherein. Evaluation of an amplification process in “real-time” generallyinvolves making periodic determinations of the amount of signalassociated with probe:amplicon complexes in the reaction mixture duringthe amplification reaction, and the determined values are used tocalculate the amount of target sequence initially present in the sample.There are a variety of methods for determining the amount of initialtarget sequence present in a sample based on real-time amplification.These include those disclosed by Light et at., “Method for Determiningthe Amount of an Analyte in a Sample,” U.S. Patent ApplicationPublication No. US 2006-0276972 (paragraphs 505 to 549); Lee et al.,“Methods for Quantitative Analysis of a Nucleic Acid AmplificationReaction,” U.S. Patent Application Publication No. US 2006-0286587;Carrick et al., “Method and Algorithm for Quantitating Polynucdeotides,”U.S. Patent Application Publication No. US 2006-0292619; Wittwer et a.,“Method for Quantification of an Analyte,” U.S. Pat. No. 6,303,305; andYokoyama et al., “Method for Assaying Nucleic Acid,” U.S. Pat. No.6,541,205. (Each of these references or the indicated portion is herebyincorporated by reference herein.) Another method for determining thequantity of target sequence initially present in a sample, but which isnot based on a real-time amplification, is disclosed by Ryder et al.,“Method for Determining Pre-Amplification Levels of a Nucleic AcidTarget Sequence from Post-Amplification Levels of Product,” U.S. Pat.No. 5,710,029, the contents of which are hereby incorporated byreference herein. The present invention is particularly suited toreal-time evaluation, because the production of side-products isdecreased, diminished, or substantially eliminated.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of example, “molecular torches” are a type ofself-hybridizing probe which includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domains that are from 1 to about 10 bases in length and areaccessible for hybridization to a target sequence present in anamplification product under strand displacement conditions. Thesingle-stranded region may be, for example, a terminal region or aninternal region, such as a loop region. Alternatively, the stranddisplacement conditions may cause “breathing” in a double-strandedterminal region of the molecular torch, thereby resulting in a transientsingle-stranded region of the terminal region which is accessible forhybridization to the target sequence. Under strand displacementconditions, hybridization of the two complementary regions (which may befully or partially complementary) of the molecular torch is favored,except in the presence of the target sequence, which will bind to thesingle-stranded region present in the target binding domain and displaceall or a portion of the target closing domain. The target binding domainand the target closing domain of a molecular torch include a detectablelabel or a pair of interacting labels (e.g., luminescent/quencher)positioned so that a different signal is produced when the moleculartorch is self-hybridized than when the molecular torch is hybridized tothe target sequence, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized moleculartorches. Molecular torches and a variety of types of interacting labelpairs are disclosed by Becker et al., “Molecular Torches,” U.S. Pat. No.6,534,274, the contents of which are hereby incorporated by referenceherein.

Another example of a detection probe exhibiting self-complementarity isa “molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complement sequence, an affinity pair (or nucleic acidarms) holding the probe in a closed conformation in the absence of atarget sequence present in an amplification product, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed by Tyagi et al., “Detectably Labeled Dual ConfirmationOligonucleotide Probes, Assays and Kits,” U.S. Pat. No. 5,925,517, andTyagi et al., “Nucleic Acid Detection Probes Having Non-FRETFluorescence Quenching and Kits and Assays Including Such Probes,” U.S.Pat. No. 6,150,097, each of which is hereby incorporated by referenceherein in its entirety.

Other self-hybridizing probes for use in the present invention are wellknown to those of ordinary skill in the art. By way of example, probebinding pairs having interacting labels, such as those disclosed byMorrison, “Competitive Homogenous Assay,” U.S. Pat. No. 5,928,862 (thecontents of which are hereby incorporated by reference herein), might beadapted for use in the present invention. Probe systems used to detectsingle nucleotide polymorphisms (snps) might also be utilized in thepresent invention. Additional detection systems include “molecularswitches,” as disclosed by Arnold et al., “Oligonucleotides Comprising aMolecular Switch,” U.S. Patent Application Publication No. US2005-0042638, the contents of which are hereby incorporated by referenceherein. And other probes, such as those comprising intercalating dyesand/or fluorochromes, might be useful for detection of amplificationproducts in the present invention. See, e.g., Ishiguro et al., “Methodof Detecting Specific Nucleic Acid Sequences,” U.S. Pat. No. 5,814,447,the contents of which are hereby incorporated by reference herein.

In those methods of the present invention where the initial targetsequence and the RNA transcription product share the same sense, it maybe desirable to initiate amplification before adding probe for real-timedetection. Adding probe prior to initiating an amplification reactionmay slow the rate of amplification since probe which binds to theinitial target sequence has to be displaced or otherwise remove duringthe primer extension step to complete a primer extension product havingthe complement of the target sequence. The initiation of amplificationis judged by the addition of amplification enzymes (e.g., a reversetranscriptase and an RNA polymerase).

In addition to the methods described herein, the present invention isdrawn to kits comprising one or more of the reagents required forcarrying out the methods of the present invention. Kits comprisingvarious components used in carrying out the present invention may beconfigured for use in any procedure requiring amplification of nucleicacid target molecules, and such kits can be customized for variousdifferent end-users. Suitable kits may be prepared, for example, forblood screening, disease diagnosis, infection control, environmentalanalysis, criminal investigations or other forensic analyses, geneticanalyses, archeological or sociological analyses, or for generallaboratory use. Kits of the present invention provide one or more of thecomponents necessary to carry out nucleic acid amplifications accordingto the invention. Kits may include reagents suitable for amplifyingnucleic acids from one particular target or may include reagentssuitable for amplifying multiple targets. Kits of the present inventionmay further provide reagents for real-time detection of one or morenucleic acid targets in a single sample, for example, one or moreself-hybridizing probes as described above. Kits may comprise a carrierthat may be compartmentalized to receive in close confinement one ormore containers such as vials, test tubes, wells, and the like.Preferably at least one of such containers contains one or morecomponents or a mixture of components needed to perform theamplification methods of the present invention.

A kit according to the present invention can include, for example, inone or more containers, a priming oligonucleotide, a promoteroligonucleotide modified to prevent primer extension by a DNA polymerase(e.g., modified to include a 3′-blocking moiety), a binding molecule orother means for terminating a primer extension reaction, and,optionally, a displacer oligonucleotide, an extender oligonucleotideand/or a capping oligonucleotide as described herein. If real-timedetection is used, the one or more containers may include one or morereagents for real-time detection of at least one nucleic acid targetsequence in a single sample, for example, one or more self-hybridizingprobes as described above. Another container may contain an enzymereagent, for example a mixture of a reverse transcriptase (either withor without RNAse H activity), an RNA polymerase, and optionally anadditional selective RNAse enzyme. (If included, the displaceroligonucleotide may be provided in the container containing the enzymereagent.) These enzymes may be provided in concentrated form or atworking concentration, usually in a form which promotes enzymestability. The enzyme reagent may also be provided in a lyophilizedform. See Shen et al., “Stabilized Enzyme Compositions for Nucleic AcidAmplification,” U.S. Pat. No. 5,834,254, the contents of which arehereby incorporated by reference herein. Another one or more containersmay contain an amplification reagent in concentrated form, e.g., 10×,50×, or 100×, or at working concentration. An amplification reagent willcontain one or more of the components necessary to run the amplificationreaction, e.g., a buffer, MgCl₂, KCl, dNTPs, rNTPs, EDTA, stabilizingagents, etc. Certain of the components, e.g., MgCl₂ and rNTPs, may beprovided separately from the remaining components, allowing the end userto titrate these reagents to achieve more optimized amplificationreactions. Another one or more containers may include reagents fordetection of amplification products, including one or more optionallylabeled detection probes. In some embodiments, a kit of the presentinvention will also include one or more containers containing one ormore positive and negative control target nucleic acids which can beutilized in amplification experiments in order to validate the testamplifications carried out by the end user. In some instances, one ormore of the reagents listed above may be combined with an internalcontrol. It is also possible to combine one or more of these reagents ina single tube or other containers.

Supports suitable for use with the invention (e.g., test tubes,multi-tube units, multi-well plates, cuvettes, flexible containers,microfluidic devices, including analytical cards or discs for use incentrifugal analyzers, etc.) may also be supplied with reagents of theinvention. Finally, a kit of the present invention may include one ormore instruction manuals provided in written or electronic form,including CD-ROMs, DVDs and video tapes. Kits of the invention maycontain virtually any combination of the components set out above ordescribed elsewhere herein. As one skilled in the art would recognize,the components supplied with kits of the invention will vary with theintended use for the kits, and the intended end user. Thus, kits may bespecifically designed to perform various functions set out in thisapplication and the components of such kits will vary accordingly.

The present invention is further drawn to various oligonucleotides,including the priming oligonucleotides, promoter oligonucleotides,terminating oligonucleotides, displacer oligonucleotides, extenderoligonucleotides, capping oligonucleotides and detection probesdescribed herein. It is to be understood that the oligonucleotides ofthe present invention may be DNA, RNA, DNA:RNA chimerics and analogsthereof, and, in any case, the present invention includes RNAequivalents of DNA oligonucleotides and DNA equivalents of RNAoligonucleotides. Except for the preferred priming oligonucleotides(including displacer oligonucleotides) and detection probes describedsupra, the oligonucleotides described hereinabove preferably comprise ablocking moiety at their 3′-termini.

Detection probes of the present invention may be labeled in a number ofalternative ways, e.g., with radioactive isotopes, fluorescent labels,chemiluminescent labels, nuclear tags, bioluminescent labels,intercalating dyes, or enzyme labels. The detection probes may includegroups of interacting labels which emit a detectable change in signalwhen the probes are hybridized to a target sequence or its complement.In various embodiments, these labeled probes optionally or preferablyare synthesized to include at least one modified nucleotide, e.g., a2′-O-methyl ribonucleotide; or these labeled oligonucleotide probesoptionally or preferably are synthesized entirely of modifiednucleotides, e.g., 2′-O-methyl ribonucleotides.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andcompositions described herein are readily apparent from the descriptionof the invention contained herein in view of information known to theordinarily skilled artisan, and may be made without departing from thescope of the invention or any embodiment thereof Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the invention. It is believed that these examplesaccurately reflect the details of experiments actually performed,however, it is possible that some minor discrepancies may exist betweenthe work actually performed and the experimental details set forth belowwhich do not affect the conclusions of these experiments. Skilledartisans will appreciate that these examples are not intended to limitthe invention to the specific embodiments described therein.Additionally, those skilled in the art, using the techniques, materialsand methods described herein, could easily devise and optimizealternative amplification systems for detecting and/or quantifying anytarget sequence.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, recombinantDNA, and chemistry, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed.,Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I andII (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gaited.,1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. See e.g., Carruthers, et al., 154 Methods inEnzymology, 287 (1987), the contents of which are hereby incorporated byreference herein. Unless otherwise stated herein, modified nucleotideswere 2′-O-methyl ribonucleotides, which were used in the synthesis astheir phosphoramidite analogs. Applicant prepared the oligonucleotidesusing an Expedite™ 8909 DNA Synthesizer (PerSeptive Biosystems,Framingham, Mass.).

Various reagents are identified in the examples below, which include anamplification reagent, an enzyme reagent, a hybridization reagent, aselection reagent, and detection reagents. Unless otherwise indicated,the formulations and pH values (where relevant) of these reagents wereas follows.

Amplification Reagent. The “Amplification Reagent” comprised 11.6 mMTrizma® base buffer, 15 mM Trizma® hydrochloride buffer, 22.7 mM MgCl₂,23.3 mM KCl, 3.33% (v/v) glycerol, 0.05 mM zinc acetate, 0.665 mM dATP,0.665 mM dCTP, 0.665 mM dGTP, 0.665 mM dTTP, 0.02% (v/v) ProClin 300Preservative (Supelco, Bellefonte, Pa.; Cat. No. 48126), 5.32 mM ATP,5.32 mM CTP, 5.32 mM GTP, 5.32 mM UTP, and 6 M HCl to pH 7.81 to 8.0 at22° C.

Enzyme Reagent. The “Enzyme Reagent” comprised 70 mMN-acetyl-L-cysteine, 10% (v/v) TRITON® X-102 detergent, 16 mM HEPES, 3mM EDTA, 0.05% (w/v) sodium azide, 20 mM Trizma® base buffer, 50 mM KCl,20% (v/v) glycerol, 150 mM trehalose, 4M NaOH to pH 7, and containing224 RTU/μL Moloney murine leukemia virus (“MMLV”) reverse transcriptaseand 140 U/μL T7 RNA polymerase, where one RTU of RT activityincorporates 1 nmol of dTMP into DE81 filter-bound product in 20 minutesat 37° C. using poly(rA)-p(dT)₁₂₋₁₈ as the substrate, and one U of T7RNA polymerase activity produces 5 fmol of RNA transcript in 20 minutesat 37° C.

Hybridization Reagent. The “Hybridization Reagent” comprised 100 mMsuccinic acid, 2% (w/v) lithium lauryl sulfate, 230 mM LiOH, 15 mMaldrithiol-2, 1.2 M LiCl, 20 mM EDTA, 20 mM EGTA, 3.0% (v/v) ethylalcohol, and 2M LiOH to pH 4.7.

Selection Reagent. The “Selection Reagent” comprised 600 mM H₃BO₃, 182mM NaOH, 1% (v/v) TRITON® X-100 detergent, and 4 M NaOH to pH 8.5.

Detection Reagent I. “Detection Reagent I” comprised 1 mM HNO₃ and 30 mMH₂O₂.

Detection Reagent II. “Detection Reagent II” comprised 1 M NaOH and 2%(w/v) Zwittergent® 3-14 detergent.

Oil Reagent: The “Oil Reagent” comprised a silicone oil (United ChemicalTechnologies, Inc., Bristol, Pa.; Cat. No. PS038).

Example 1 Comparison of Blocked and Unblocked Promoter Oligonucleotides

This experiment was conducted to evaluate the specificity of anamplification method according to the present invention in which aregion (“the target region”) of a cloned transcript derived from the 5′untranslated region of the hepatitis C virus (“the transcript”) wastargeted for amplification. For this experiment we prepared two sets ofpriming and promoter oligonucleotides having identical base sequences.The two sets of oligonucleotides differed by the presence or absence ofa 3′-terminal blocking moiety on the promoter oligonucleotide. Thepromoter oligonucleotide in each set targeted the complement of asequence contained within the 5′-end of the target region and had thebase sequence of SEQ ID NO:5 aatttaatacgactcactatagggagactagccatggcgttagtatgagtgtcgtgcag, where the underlined portion of the promoteroligonucleotide constitutes a T7 promoter sequence (SEQ ID NO:3) and thenon-underlined portion represents a hybridizing sequence (SEQ ID NO:4).The priming oligonucleotide in each set targeted a sequence containedwithin the 3′-end of the target region and had the base sequence of SEQID NO:6. Also included in the amplification method was a terminatingoligonucleotide made up of 2′-O-methyl ribonucleotides having the basesequence of SEQ ID NO:38 ggcuagacgcuuucugcgugaaga. The terminatingoligonucleotide had a 3′-terminal blocking moiety and targeted a regionof the transcript just 5′ to the target region. The 5′-ends of theterminating oligonucleotide and of the hybridizing sequence of thepromoter oligonucleotide overlapped by six bases. The 3′-terminalblocking moiety of both the promoter oligonucleotide and the terminatingoligonucleotide consisted of a 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Sterling, Va.; Cat. No.20-0102-01).

For amplification, 75 μL of the Amplification Reagent was added to eachof eight reaction tubes. The Amplification Reagent was then combinedwith 30 pmol of a promoter oligonucleotide, 30 pmol of the primingoligonucleotide and 5 pmol of the terminating oligonucleotide. One setof four of the tubes was provided with 30 pmol of the unblocked promoteroligonucleotide (group I), and another set of four tubes was providedwith 30 pmol of the blocked promoter oligonucleotide (group II). Next, 1μL of a 0.1% (w/v) lithium lauryl sulfate (“LLS”) buffer containing 1000copies/μL of the transcript was added to two of the tubes in each group,while the remaining two tubes in each group served as negative controls.The reaction mixtures were overlaid with 200 μL of the Oil Reagent, andthe tubes were then sealed and hand-shaken horizontally for 5 to 10seconds before the tubes were incubated in a 60° C. water bath for 10minutes. The tubes were then transferred to a 41.5° C. water bath andincubated for 15 minutes before adding 25 μL of the Enzyme Reagent toeach tube. After adding the Enzyme Reagent, the tubes were sealed,removed from the water bath and hand-shaken horizontally for 5 to 10seconds to fully mix the components of the reaction mixtures. The tubeswere returned to the 41.5° C. water bath and incubated for an additional60 minutes to facilitate amplification of the target region in thepresence of MMLV reverse transcriptase and T7 RNA polymerase. Followingamplification, the tubes were removed from the 41.5° C. water bath andallowed to cool at room temperature for 10 to 15 minutes.

A 5 μL aliquot of each reaction mixture was taken from the tubes,diluted 1:1 with a 2× Novex® TBE-Urea Sample Buffer (InvitrogenCorporation, Carlsbad, Calif.; Cat. No. LC6876), and loaded onto aNovex® TBE-Urea Denaturing Gel (Invitrogen; Cat. No. EC6865BOX). The gelwas held by an Xcell Surelock™ Mini-Cell (Invitrogen; Cat. No. EI0001)and run at 180 volts for 50 minutes using a 5× Novex® TBE Running Buffer(Invitrogen; Cat. No. LC6675) diluted 1:4 with deionized water.Afterwards, the gel was stained with 0.5 μg/mL of ethidium bromide in a1× TBE (Tris-Borate-EDTA) solution, visualized on a FisherBiotech®Ultraviolet Transilluminator (Fisher Scientific International Inc.,Hampton, N.H.; Model No. FB-TIV-816A), and photographed with a handheldcamera using Polaroid 667 film.

The results of this experiment are illustrated in the photographed gelof FIG. 3, Each number above the pictured gel represents a distinctlane, where lane 1 is an RNA ladder of 100, 200, 300, 400, 500, 750 and1000 base oligonucleotides, and the remainder of the lanes correspond tothe following reaction mixtures: (i) lanes 2 and 3 correspond to thetranscript-containing replicates of group I (unblocked promoteroligonucleotide); (ii) lanes 4 and 5 correspond to thetranscript-containing replicates of group II (blocked promoteroligonucteotide); (iii) lanes 6 and 7 correspond to thetranscript-negative replicates of group I; and (iv) lanes 8 and 9correspond to the transcript-negative replicates of group II. The firstvisible band in lanes 2-5 constitutes amplicon derived fromamplification of the target region. The remainder of the bands in lanes2, 3, 6 and 7 constitute non-specific amplification products. Thus, theresults indicate that only amplification using the fully blockedpromoter oligonucleotides was specific, as there was no visibleside-product formation in either the transcript-containing or transcriptnegative reaction mixtures containing blocked promoter oligonucleotides,whereas visible side-products were formed in both thetranscript-containing and transcript-negative reaction mixturescontaining unblocked promoter oligonucleotides.

Example 2 Reduction in the Formation of Replicating Molecules

This experiment was designed to evaluate whether the use of a blockedpromoter oligonucleotide in an amplification method of the presentinvention would lead to a reduction in the formation of replicatingmolecules over a standard transcription-based amplification procedure.Replicating molecules are generally believed to form when the 3′-end ofa promoter oligonucleotide forms a hairpin structure and is extended inthe presence of a polymerase, thereby forming a double-stranded promotersequence. Transcription initiated from the double-stranded promotersequence results in the formation of amplicon containing an antisenseversion of the promoter sequence.

In this experiment, we compared the production of replicating moleculesin amplification reactions containing promoter oligonucleotides thatwere either blocked or unblocked at their 3′-terminal ends in thepresence or absence of purified rRNA from Mycobacterium tuberculosis(ATCC No. 25177) using one of two detection probes targeting a region(“the target region”) of the 16S rRNA of Mycobacterium tuberculosis(“the target nucleic acid”). The blocked and unblocked promoteroligonucleotides targeted sequences contained within the complement ofthe 5′-end of the target region. The blocked promoter oligonucleotidehad the base sequence of SEQ ID NO:26aattctaatacgactcactatagggagaactgggtctaataccggataggaccacgggatgcat, andthe unblocked promoter oligonucleotide had the base sequence of SEQ IDNO:28 aattctaatacgactcactatagggagaactgggtctaat accggataggaccacggga,where the underlined portion of each promoter oligonucleotideconstitutes a T7 promoter sequence (SEQ ID NO:3) and the non-underlinedportion represents a hybridizing sequence (SEQ ID NO:25 and SEQ IDNO:27). The priming oligonucleotide targeted a sequence contained withinthe 3′-end of the target region and had the base sequence of SEQ IDNO:29. Also included was a terminating oligonucleotide made up of2′-O-methyl ribonucleotides having the base sequence of SEQ ID NO:39cccaguuucccaggcuuauccc. The terminating oligonucleotide targeted aregion of the target nucleic acid just 5′ to the target region and had a3′-terminal blocking moiety. The 5′-ends of the terminatingoligonucleotide and the hybridizing sequence of the promoteroligonucleotide overlapped by six bases. The 3′-terminal blocking moietyof both the blocked promoter oligonucleotide and the terminatingoligonucleotide consisted of the 3′- to-3′ linkage described inExample 1. And for detection, two detection probes were synthesized. Thefirst detection probe (“detection probe I”) comprised 2′-O-methylribonucleotides targeted a sequence contained within the target regionand had the base sequence of SEQ ID NO:30 gcucauccca*caccgcuaaagc. Thesecond detection probe (“detection probe II”) targeted the antisense ofa region contained within the T7 promoter sequence and had the basesequence of SEQ ID NO:40 atacgactc*actata. The asterisk in bothdetection probe sequences indicates the position of a4-(2-succinimidyloxycarbonylethyl)-phenyl-10-methylacridinium-9-carboxylate fluorosulfonateacridinium ester label (“standard AE”) joined to the probe by means of anon-nucleotide linker, as described by Arnold et al., “Linking Reagentsfor Nucleotide Probes,” U.S. Pat. No. 5,585,481, the contents of whichare hereby incorporated by reference herein.

A total of eight different amplification reactions were performed inreplicates of five. All of the reaction tubes used for the amplificationreactions were provided with 75 μL of the Amplification Reagent,followed by 5 pmol each of either the blocked or unblocked promoteroligonucleotide, the priming oligonucleotide, and the terminatingoligonucleotide. Two sets of the tubes received 2 μL each of a 0.2%(w/v) LLS buffer containing 250 copies/μL of the target nucleic acid,and the other two sets of tubes received no target nucleic acid. Thereaction mixtures were overlaid with 200 μL of the Oil Reagent, and thetubes were then sealed and hand-shaken horizontally for 5 to 10 secondsbefore being incubated in a 60° C. water bath for 10 minutes. The tubeswere then transferred to a 41.5° C. water bath and incubated for 10minutes before adding 25 μL of the Enzyme Reagent to each tube. Afteradding the Enzyme Reagent, the tubes were sealed, removed from the waterbath and hand-shaken horizontally for 5 to 10 seconds to fully mix thecomponents of the reaction mixtures. The tubes were returned to the41.5° C. water bath and incubated for an additional 60 minutes to permitamplification of the target sequences. Following amplification, thetubes were removed from the 41.5° C. water bath and allowed to cool atroom temperature for 10 to 15 minutes.

The detection step was performed in accordance with the HybridizationProtection Assay disclosed by Arnold et al., “Homogenous ProtectionAssay,” U.S. Pat. No. 5,283,174. In this step, 100 μL of theHybridization Reagent containing either 52 fmol of detection probe I or10.2 fmol of detection probe II was added to each tube. After adding thedetection probes, the tubes were sealed, hand-shaken horizontally for 5to 10 seconds, and incubated in a 60° C. water bath for 15 minutes topermit hybridization of the detection probes to their correspondingtarget sequences. Following hybridization, 250 μL of the SelectionReagent was added to the tubes and the tubes were sealed and hand-shakenhorizontally for 5 to 10 seconds before being incubated in a 60° C.water bath for 10 minutes to hydrolyze acridinium ester labelsassociated with unhybridized probe. The samples were cooled at roomtemperature for at least 10 minutes before being analyzed in a LEADER®HC+ Luminometer (Gen-Probe Incorporated, San Diego, Calif.; Cat. No.4747) equipped with automatic injection of Detection Reagent I, followedby automatic injection of Detection Reagent II. Signal emitted from thetubes was measured in relative light units (“RLU”), which is a measureof chemiluminescence.

The results were averaged for each set of reaction conditions and arepresented in Table 1 below. From these results, it can be seen thatthose amplification reactions containing the blocked promoteroligonucleotide performed as well as those amplification reactionscontaining the unblocked promoter oligonucleotide at amplifying atargeted region of the target nucleic acid. However, those amplificationreactions containing the blocked promoter oligonucleotide producedsubstantially fewer replicating molecules than did those amplificationreactions containing the unblocked promoter oligonucleotide, both in thepresence and in the absence of the transcript.

TABLE 1 Effect of 3′-Blocking Promoter Oligonucleotides on the Formationof Replicating Molecules Detection Probe I Detection Probe II Target NoTarget Target No Target Nucleic Nucleic Nucleic Nucleic Acid Acid AcidAcid Blocked Promoter 1,047,084 4,222 64,874 10,063 OligonucleotideUnblocked Promoter 976,156 98,067 526,657 456,130 Oligonucleotide

Example 3 Sensitivity of Amplification Assay Using Blocked PromoterOligonucleotide and Terminating Oligonucleotide

This experiment examined the sensitivity of an amplification systemaccording to the present invention in which a region (“the targetregion”) of purified 23S rRNA from Chlamydia trachomatis (ATCC No.VR-878) (“the target nucleic acid”) was targeted for amplification.Included in this experiment was a promoter oligonucleotide having a3′-terminal blocking moiety, a priming oligonucleotide, a terminatingoligonucleotide having a 3′-terminal blocking moiety, and a labeleddetection probe. The promoter oligonucleotide targeted the complement ofa sequence contained within the 5′-end of the target region and had thebase sequence of SEQ ID NO:22aatttaatacgactcactatagggagacggagtaagttaagcacgcggac gattgga, where theunderlined portion of the promoter oligonucleotide constitutes a T7promoter sequence (SEQ ID NO:3) and the non-underlined portionrepresents a hybridizing sequence (SEQ ID NO:21). The primingoligonucleotide targeted a sequence contained within the 3′-end of thetarget region and had the base sequence of SEQ ID NO:23. The terminatingoligonucleotide was made up of 2′-O-methyl ribonucleotides having thebase sequence of SEQ ID NO:41 uccgucauuccuucgcuauagu and targeted aregion of the target nucleic acid just 5′ to the target region. The5′-ends of the terminating oligonucleotide and the hybridizing sequenceof the promoter oligonucleotide overlapped by four bases. The3′-terminal blocking moiety of both the promoter oligonucleotide and theterminating oligonucleotide consisted of the 3′-to-3′ linkage describedin Example 1. The detection probe targeted a sequence contained withinthe target region and was made up of 2′-O-methyl ribonucleotides havingthe base sequence of SEQ ID NO:24 eguucucaucgcueu*acggacucu, where theasterisk indicates the position of a standard AE label joined to theprobe by means of a non-nucleotide linker. See Arnold et al., U.S. Pat.No. 5,585,481.

Amplification in this experiment was carried out essentially asdescribed in Example 1. Each amplification reaction was performed inreplicates of 3, and the target nucleic acid was added to each reactiontube in each set of replicates in copy numbers of 10, 100, 1000 or10,000 from a 0.1% (w/v) LLS buffer containing 10, 100, 1000 or 10,000copies/μL, respectively. The promoter and priming oligonucleotides wereeach added to the tubes in 30 pmol/reaction amounts, and 5 pmol of theterminating oligonucleotide was added to each tube. Using the Chlamydiatrachomatis probe of this experiment, detection was carried outessentially as described in Example 2. The results of this experimentare set forth in Table 2 below and indicate 100 copy sensitivity forthis amplification system, where an average RLU value of above 10,000constituted a positive result.

TABLE 2 Sensitivity of Chlamydia trachomatis Amplification System CopyNumber Avg. RLU 10 8504 100 51,574 1000 1,578,416 10,000 6,092,697

Example 4 Amplification of a Double-Stranded Target Sequence

This example examines an amplification system according to the presentinvention in which a region (“the target region”) of a cloned,double-stranded transcript derived from the E6 and E7 genes of humanpapilloma virus type 16 (“HPV-16”) (“the transcript”) was targeted foramplification. See FIG. 1C. This experiment included a promoteroligonucleotide having a 3′-terminal blocking moiety, a primingoligonucleotide and a labeled detection probe. The promoteroligonucleotide targeted the complement of a sequence contained withinthe 5′-end of the target region and had the base sequence of SEQ IDNO:14 aatttaatacgactcactatagggagagaacagatggggcacacaattcctagt, where theunderlined portion of the promoter oligonucleotide constitutes a T7promoter sequence (SEQ ID) NO:3) and the non-underlined portionrepresents a hybridizing sequence (SEQ ID NO:13). The 3′-terminalblocking moiety of the promoter oligonucleotide consisted of the3′-to-3′ linkage described in Example 1. The priming oligonucleotidetargeted a sequence contained within the 3′-end of the target region andhad the base sequence of SEQ ID NO:15. The detection probe, which wascomprised of 2′-O-methyl ribonucleotides, had the base sequence of SEQID NO:16 ggacaa*gcagaaccggaea and targeted a sequence contained withinthe target region. The asterisk indicates the position of a standard AElabel joined to the probe by means of a non-nucleotide linker. SeeArnold et al., U.S. Pat. No. 5,585,481.

The amplification reactions of this experiment were performed inreplicates of 5, and each tube included 75 μL of the AmplificationReagent containing 0, 50, 100, 500, 1000 or 5000 copies of thetranscript. Each tube was also provided with 40 pmol of the promoteroligonucleotide and 15 pmol of the priming oligonucleotide. The reactionmixtures were overlaid with 200 μL of the Oil Reagent, and the tubeswere then sealed and hand-shaken horizontally for 5 to 10 seconds. Toseparate the complementary strands of the double-stranded transcript,the tubes were incubated in a heat block for 10 minutes at 95° C. At theend of this incubation, the tubes were removed from the heat block andrapidly cooled on ice for 5 minutes to promote association of thepriming oligonucleotide and the targeted region of the transcript. Thetubes were then incubated in a 41.5° C. water bath for 10 minutes. Toinitiate amplification, 25 μL of the Enzyme Reagent was added to thetubes, which were then sealed and hand-shaken horizontally for 5 to 10seconds to fully mix the Amplification and Enzyme Reagents.Amplification was then carried out by returning the tubes to the 41.5°C. water bath for a 1 hour incubation.

Following amplification, detection of the amplification products wasperformed in the manner described in Example 2 using 100 fmol/reactionof the detection probe. The results of this experiment are set forth inTable 3 below and indicate 500 copy sensitivity for this amplificationsystem, where an RLU value of 10,000 or greater constituted a positiveresult.

TABLE 3 Sensitivity of HPV-16 Amplification System % Positive CopyNumber Avg. RLU Amplifications 0 5410 0 50 5647 0 100 6018 0 500 19,92880 1000 200,072 80 5000 371,641 100

Example 5 Comparison of Blocked and Unblocked Promoter Oligonucleotides

The purpose of this experiment was to evaluate the benefit of includinga terminating oligonucleotide in the HCV amplification system ofExample 1. See FIG. 1A. For this experiment, four different reactionmixtures were set up in replicates of 10 containing either 0 or 10copies of the transcript of Example 1 in the presence or absence of aterminating oligonucleotide. The promoter, priming and terminatingoligonucleotides were identical to those used in Example 1. UnlikeExample 1, this experiment included two detection probes, both of whichwere made up of 2′-O-methyl ribonucleotides and targeted a sequencecontained within the region of the transcript targeted foramplification. The first detection probe had the base sequence of SEQ IDNO:7 guacu*caccgguucc, and the second detection probe had the basesequence of SEQ ID NO:8 agaccacua*uggcucucccggg. Each detection probehad a “cold,” or unlabeled version, and a “hot,” or labeled version.(Cold probes were used in this experiment to prevent saturation of thehot probes in the presence of a vast excess of amplicon, therebypermitting the extent of amplification to be evaluated.) The asterisksindicate the positions of standard AU labels joined to the hot probes bymeans of non-nucleotide linkers. See Arnold et al., U.S. Pat. No.5,585,481.

The amplification reactions were essentially carried out in the mannerdescribed in Example 2 using 30 pmol/reaction of the promoteroligonucleotide and 15 pmol/reaction of each of the priming andterminating oligonucleotides. Detection was performed as described inExample 2 using 100 fmol/reaction of each of the two hot probes and eachof the two cold probes in amounts corresponding to the ratios indicatedin Table 4 below. The averaged results are set forth in Table 4 inrelative light units (“RLU”) and demonstrate that only those reactionmixtures containing the terminating oligonucleotide had 10 copy levelsensitivity in the HCV amplification system. The coefficient ofvariation values (“% CV”) appearing in Table 4 for the different copylevels tested constitute the standard deviation of the replicates overthe mean of the replicates as a percentage.

TABLE 4 Sensitivity of the HCV Amplification System in the Presence andAbsence of a Terminating Oligonucleotide Copy Terminating Cold Prot/HotNumber Oligonucleotide Probe Ratio Avg. RLU % CV 0 + 25:1 15,813 7.510 + 25:1 635,695 83.5 0 −  5:1 15,378 14.3 10 −  5:1 20,730 37.5

Example 6 Varying Length of Base Overlap Between PromoterOligonucleotide and Terminating Oligonucleotide

In this experiment, we studied the effect of varying the length ofoverlap between a blocked promoter oligonucleotide and a terminatingoligonucleotide on amplification efficiency in the HCV amplificationsystem of Example 1. The reaction mixtures were set up in replicates offour and each set was provided with 0 or 50 copies of the transcript ofExample 1. The amount of overlap between the promoter oligonucleotideand the terminating oligonucleotide, if present, was 2, 4 or 6 bases foreach set of reaction mixtures. The promoter oligonucleotide, the primingoligonucleotide, and the detection probes were identical to those usedin Example 5. The cold probes and hot probes were used at a ratio of4:1. The three terminating oligonucleotides of this experiment were madeup of 2′-O-methyl ribonucleotides and had the following base sequences:(i) SEQ ID NO:42 agacgcuuucugcgugaagacagu (2 base overlap); (ii) SEQ IDNO:43 cuagacgcuuucugcgugaagaca (4 base overlap); and (iii) SEQ ID NO:38(6 base overlap).

The amplification reactions were carried out in reaction tubes in themanner described in Example 5 using 30 pmol/reaction of the promoteroligonucleotide and 15 pmol/reaction each of the priming oligonucleotideand the terminating oligonucleotides. Detection was performed asdescribed in Example 2 using 100 fmol/reaction of each of the two hotprobes and 400 fmol/reaction of each of the two cold probes. Theaveraged results are set forth in Table 5 in relative light units(“RLU”) and indicate that under the conditions tested, six bases ofoverlap between the promoter oligonucleotide and the terminatingoligonucleotide is optimal for the HCV amplification system. The skilledartisan could apply this method to any amplification system to determinethe optimal amount of overlap between a promoter oligonucleotide and aterminating oligonucleotide using nothing more than routineexperimentation.

TABLE 5 Effect of Terminating Oligonucleotide/Promoter OligonucleotideBase Overlap on Amplification Efficiency Copy Terminating Base NumberOligonucleotide Overlap Avg. RLU 0 − N/A 29,593 + 2 25,430 + 4 27,128 +6 27,732 50 − N/A 265,250 + 2 339,833 + 4 253,577 + 6 1,904,911

Example 7 Comparison of Real-Time Amplification Assays in the Presenceor Absence of a Terminating Oligonucleotide

This experiment was conducted to determine whether a terminatingoligonucleotide improves amplification performance in a real-timeamplification assay. For this experiment, we used the Mycobacteriumtuberculosis amplification system of Example 2, which included theunblocked promoter oligonucleotide having the base sequence of SEQ IDNO:28, the priming oligonucleotide having the base sequence of SEQ IDNO:29, and the blocked terminating oligonucleotide having the basesequence of SEQ ID NO:39. Also included was a molecular beacon detectionprobe having the base sequence of SEQ ID NO:31. The detection probe wassynthesized to include a BHQ-2 Black Hole Quencher™ Dye joined to its3′-end using a BHQ-2 Glycolate CPG (Biosearch Technologies, Inc.,Novato, Calif.; Cat. No. CG5-5042G-1) and a Cy™ 5 Dye joined to its5′-end using a Cy™ 5-CE phosphoramidite (Glen Research; Cat No.105915-90). The reactions were run in the wells of a Thermo LabsystemsWhite Cliniplate 96 (VWR International, Inc., West Chester, Pa.; Cat.No. 28298-610), and each reaction well contained 0, 100 or 1000 copiesof the target nucleic acid of Example 2. For each copy number tested,there were four replicates which included the terminatingoligonucleotide and four replicates which did not.

For amplification and detection, 75 μL of the Amplification Reagent wasadded to each reaction well, followed by the addition of 2 μL of a 0.1%(w/v) LLS buffer containing 50 copies/μL to each tube of one set ofreplicates and 2 μL of a 0.1% (w/v) LLS buffer containing 500 copies/μLto each tube of another set of replicates. The promoter oligonucleotide,the priming oligonucleotide and, when included, the terminatingoligonucleotide were each added to the tubes in 5 pmol/reaction amounts,and 2 pmol/reaction of the detection probe was added to each tube.Target nucleic acid was provided to the reaction wells in the amountsindicated, and the reactions mixtures were overlaid with 80 μL of theOil Reagent. The plate was sealed with a ThermalSeal RT™ Film(Sigma-Aldrich Corporation, St. Louis, Mo.; Product No. Z369675) and thecontents of the plate were subjected to a 60° C. incubation for 15minutes in a Solo HT Microplate Incubator (Thermo Electron Corporation,Waltham, Mass.; Model No. 5161580), followed by a 42° C. incubation for10 minutes in the Solo HT Microplate Incubator. Next, 25 μL of theEnzyme Reagent (pre-heated to 42° C.) was added to each well and thecontents were mixed several times using a pipette. The contents of theplate were then incubated at 42° C. for 120 minutes in a Biolumin™ 960Micro Assay Reader (Molecular Dynamics Inc., Sunnyvale, Calif.) andfluorescence from the Cy™ 5 Dye channel was monitored as a function oftime in one minute intervals. The results of this monitoring, which aregraphically presented in FIGS. 4A-F, indicate that the terminatingoligonucleotide dramatically enhanced amplification of the targetsequence in the Mycobacterium tuberculosis real-time amplificationassay.

Example 8 Terminating Oligonucleotides Versus Digestion Oligonucleotides

This experiment compared levels of amplification in the Mycobacteriumtuberculosis amplification system of Example 2 using either aterminating oligonucleotide or a digestion oligonucleotide in thepresence of a blocked or unblocked promoter oligonucleotide. Theterminating oligonucleotide of this experiment was designed to bind tothe targeted RNA and physically block the activity of the reversetranscriptase enzyme, while the digestion oligonucleotide, which wascomposed of DNA, was designed to bind to the targeted RNA and directdigestion of the substrate RNA by an RNAse H activity. Use of theterminating or digestion oligonucleotide results in the formation of atemplate-complementary strand, or cDNA, having a defined 3′-end. Thepromoter oligonucleotide is designed so that its template-bindingportion hybridizes to a 3′-terminal sequence present in thetemplate-complementary strand, thereby facilitating the formation of adouble-stranded promoter sequence in the presence of the reversetranscriptase enzyme.

As in Example 2, the promoter oligonucleotide of this experiment had thebase sequence of SEQ ID NO:28 and the priming oligonucleotide had thebase sequence of SEQ ID NO.29. The terminating oligonucleotide was madeup of 2′-O-methyl ribonucleotides having the base sequence of SEQ IDNO:44 caguuucccaggcuuauccc, and the digestion oligonucleotide had thebase sequence of SEQ ID NO:45 gtattagacccagfttcccaggct. The 5′-ends ofthe terminating oligonucleotide the hybridizing sequence of the promoteroligonucleotide identified in Example 2 overlapped by four bases, andthe first 14 bases extending from the 5′-end of the digestionoligonucleotide overlapped with the 5′-most 14 bases of the hybridizingsequence of the promoter oligonucleotide. The blocked promoteroligonucleotide, the terminating oligonucleotide, and the digestionoligonucleotide all included a 3′-terminal blocking moiety consisting ofthe 3′-to-3′ linkage described in Example 1. And the detection probe hadthe base sequence of SEQ ID NO:32 getcatccca*caccgctaaagc, where theasterisk indicates the position of a standard AE label joined to theprobe by means of a non-nucleotide linker. See Arnold et al, U.S. Pat.No. 5,585,481.

A total of six different reactions were performed in replicates of two,as set forth in Table 6 below. Template positive reactions were eachprovided with 1 μL of a 0.1% (w/v) LLS buffer containing 50 copies/μL ofthe Mycobacterium tuberculosis target nucleic acid of Example 2, andtemplate negative reactions included no target nucleic acid.Amplification and detection were essentially carried out as in Example 2using 30 pmol/reaction each of the promoter and primingoligonucleotides, 5 pmol/reaction of the terminating oligonucleotide, 30pmol/reaction of the digestion oligonucleotide, and 10 fmol/reaction ofthe detection probe. The results of these reactions, which were measuredin relative light units (“RLU”), are presented in Table 6 and indicatethat amplification in this amplification system was similar in thepresence of either the terminating or the digestion oligonucleotide,although performance was somewhat better using the digestionoligonucleotide. Additionally, the results indicate that the level ofamplification in this amplification system at this copy number wasenhanced in the presence of the digestion oligonucleotide.

TABLE 6 Amplicon Production Using Terminating or DigestionOligonucleotide Terminating (T) or Digestion (D) Promoter ReactionTemplate Oligonucleotide Oligonucleotide RLU 1 Positive T Blocked319,449 2 T Blocked 254,181 3 T Unblocked 20,915 4 T Unblocked 3767 5 DBlocked 472,786 6 D Blocked 422,818 7 D Unblocked 162,484 8 D Unblocked136,134 9 None Blocked 10,007 10 None Blocked 5052 11 Negative D Blocked27,594 12 D Blocked 5157

Example 9 Capped Priming Oligonucleotides

This experiment studied the effect of including a primingoligonucleotide cap on side-product formation using the Mycobacteriumtuberculosis amplification system of Example 2. A “cap” is a shortoligonucleotide complementary to the 3′-terminal end of a primingoligonucleotide and includes a 3′-terminal blocking moiety to preventextension from a terminal 3′-OH group. The cap is included to preventthe priming oligonucleotide from forming an oligonucleotide dimer withthe promoter oligonucleotide, which could result in the formation of afunctional double-stranded promoter sequence if the primingoligonucleotide is extended in the presence of a reverse transcriptaseenzyme. As illustrated in FIG. 5A, the formation of an oligonucleotidedimer having a functional double-stranded promoter sequence could leadto the production of unwanted side-products in the presence of an RNApolymerase. While the cap inhibits oligonucleotide dimer formation, thecap can be readily displaced from the priming oligonucleotide throughspecific hybridization with the template sequence. A diagram of capusage is shown in FIG. 6A.

For this experiment, we tested three different reaction conditions inreplicates of two in the presence or absence of the Mycobacteriumtuberculosis target nucleic acid of Example 2. The components of thethree reaction conditions differed as follows: (i) the first set ofreaction conditions included an unblocked promoter oligonucleotide, anuncapped priming oligonucleotide and a blocked terminatingoligonucleotide; (ii) the second set of reaction conditions included ablocked promoter oligonucleotide, an uncapped priming oligonucleotide,and a blocked terminating oligonucleotide; and (iii) the third set ofreaction conditions included a blocked promoter oligonucleotide, apriming oligonucleotide hybridized to a blocked cap at its 3′-terminalend, and a blocked terminating oligonucleotide. As in Example 2, thepromoter oligonucleotide had the base sequence of SEQ ID NO:28 and thepriming oligonucleotide had the base sequence of SEQ ID NO:29. The caphad the base sequence of SEQ ID NO:46 ctatc. The terminatingoligonucleotide was made up of 2′-O-methyl ribonucleotides having thebase sequence of SEQ ID NO:44 caguuucccaggcuuauccc. And the terminatingoligonucleotide, the promoter oligonucleotide, when blocked, and the capall included a 3′-terminal blocking moiety consisting of the 3′-to-3′linkage described in Example 1.

Prior to initiating amplification, the priming oligonucteotide and thecap were pre-hybridized in a 10 mM NaCl solution containing the primingoligonucleotide and the cap at a 1:1 ratio. The facilitatehybridization, the reaction tubes containing the solution were incubatedin a 95° C. water bath for 10 minutes and then cooled at roomtemperature for 2 hours. Following this pre-hybridization step,amplification was carried out as in Example 2 using 30 pmol/reactioneach of the promoter oligonucleotide and the capped primingoligonucleotide and 5 pmol/reaction of the terminating oligonucleotide,where each reaction mixture was also provided with 1 μL of a 0.1% (w/v)LLS buffer containing 10,000 copies/μL of the target nucleic acid. Afteramplification, a 5 μL sample was taken from each tube, diluted 1:1 witha 10× BlueJuice™ Gel Loading Buffer (Invitrogen; Cat. No. 10816-015)which was diluted to 2× with TBE (Tris-Borate-EDTA), and loaded onto anF-Gel® Single Comb Gel (4% high resolution agarose) which waspre-stained with ethidium bromide (Invitrogen; Cat. No. 05018-04). Thegels were run on an E-Gel® Base (Invitrogen; Cat. No. G5100-01) at 80volts for 30 minutes. The gels were then visualized on a FisherBiotech®Ultraviolet Transilluminator and photographed with a handheld camerausing Polaroid 667 film.

The results of this experiment are illustrated in the photographed gelsof FIG. 7A (template negative gel) and FIG. 7B (template positive gel).The numbers above the pictured gels indicate distinct lanes, where lane7 is blank, lane 8 is a 100 base pair RNA ladder, lane 9 is a 20 basepair RNA ladder, and the remainder of the lanes contain products fromthe following reaction mixtures: (i) lanes 1 and 2 correspond toreaction mixtures containing the unblocked promoter oligonucleotide, theuncapped priming oligonucleotide, and the blocked terminatingoligonucleotide; (ii) lanes 3 and 4 correspond to reaction mixturescontaining the blocked promoter oligonucleotide, the uncapped primingoligonucleotide, and the terminating oligonucleotide; and (iii) lanes 5and 6 correspond to reaction mixtures containing the blocked promoteroligonucleotide, the capped priming oligonucleotide, and the terminatingoligonucleotide. The results clearly show that capping the primingoligonucleotide resulted in a further reduction in side-productformation (the side-products, which are oligonucleotide dimers in thesereactions, are in the 20-mer to 60-mer range, whereas the amplicon wouldbe greater than 100 bases in length).

Example 10 Looped Priming Oligonucleotides

In this experiment, the effect of looped priming oligonucleotides onamplification in the Mycobacterium tuberculosis amplification system ofExample 2 was examined. Looped priming oligonucleotides are a variety ofthe priming oligonucleotides and caps evaluated in Example 9. A loopedpriming oligonucleotide includes a cap which is joined at its 3′-end tothe 5′-end of the priming oligonucleotide by means of a non-nucleotidelinker (e.g., a basic nucleotides). One advantage of a looped primingoligonucleotide is that reassociation of the priming oligonucleotide andthe cap, in the absence of the targeted template, is faster when the twooligonucleotides are maintained in close proximity to each other.Another advantage of a looped priming oligonucleotide is that thepriming oligonucleotide and the cap can be generated in a singlesynthesis procedure, as opposed to the time intensive syntheses ofseparate priming and cap oligonucleotides.

Comparison was made between an uncapped priming oligonucleotide andlooped priming oligonucleotides having caps of varying lengths. Thepromoter, priming and terminating oligonucleotides were the same asthose used in Example 9, and the detection probe was the same asdetection probe I used in Example 2. The detection probe was provided tothe reaction mixtures in both “cold” and “hot” forms, for the reasonsdescribed in Example 5, and the cold:hot probe ratio of each reactionmixture was 250:1. The looped priming oligonucleotides had the followingsequences, where each “n” represents an a basic nucleotide (GlenResearch; Cat. No. 10-1924-xx):

Looped Priming Oligonucleotide I (LPO I): SEQ ID NO:47ctatttnngccgtcaccccaccaaca agctgatag;

Looped Priming Oligonucleotide II (LPO II): SEQ ID NO:48ctatcnnnnngccgtcacccca ccaacaagotgatag;

Looped Priming Oligonucleotide III (LPO III): SEQ ID NO:49ctatnnnnngccgtcacccca ccaacaagotgatag;

Looped Priming Oligonucleotide IV (LPO IV): SEQ ID NO:50ctatcannnnngccgtcaccc caccaacaagctgatag;

Looped Priming Oligonucleotide V (LPO V): SEQ ID NO:51ctatcnnnngccgtcaccccac caacaagctgatag;

Looped Priming Oligonucleotide VI (LPO VI): SEQ ID NO:52ctatcannnngccgtcacccc accaacaagctgatag; and

Looped Priming Oligonucleotide VII (LPO VII): SEQ ID NO:53ctatcagcttgttggnnnnn gccgtcaccccaccaacaagctgatag.

A different reaction mixture was prepared for each primingoligonucleotide, and the reaction mixtures were tested in replicates ofthree using 1000 copies of the Mycobacterium tuberculosis target nucleicacid of Example 2 obtained from 0.1% (w/v) LLS buffer containing 1000copies/μL of the target nucleic acid. The amplification and detectionsteps were carried out as in Example 2 using 30 pmol/reaction each ofthe promoter and priming oligonucleotides, 5 pmol/reaction of theterminating oligonucleotide, 10 fmol/reaction of the hot probe, and 2.5pmol/reaction of the cold probe. Signal from the tubes was measured inrelative light units (“RLU”) and the average RLU values are presented inTable 7 below. The results indicate that the template can be amplifiedusing a looped priming oligonucleotide, and that a looped primingoligonucleotide having four a basic groups and a five base cap isoptimal for the Mycobacterium tuberculosis amplification system.

TABLE 7 Effect of Looped Priming Oligonucleotides on AmplificationPriming Oligonucleotide Avg. RLU Uncapped 430,060 LPO I 292,541 LPO II260,559 LPO III 281,304 LPO IV 136,398 LPO V 372,119 LPO VI 171,382 LPOVII 20,045

Example 11 Comparison of Looped Priming Oligonucleotides and Caps

This experiment evaluated the ability of looped priming oligonucleotidesto inhibit primer-dependent side-product formation. For this experiment,looped priming oligonucleotides LPO V and LPO VII of Example 10 werecompared with an uncapped priming oligonucleotide and a primingoligonucleotide having a 14 base cap. The uncapped and capped primingoligonucleotides were the same as the uncapped priming oligonucleotideused in Example 10, and the cap had the base sequence of SEQ ID NO:54ctatcagcttgttg (the cap and the priming oligonucleotide werepre-hybridized as in Example 9). The terminating oligonucleotide was thesame as the terminating oligonucleotide used in Example 10, and thedetection probe targeted the complement of the priming oligonucleotideand had the base sequence of SEQ ID NO:29 gccgtcacccc*accaacaagctgatag,where the asterisk indicates the position of a standard AE label joinedto the probe by means of a non-nucleotide linker. See Arnold et at.,U.S. Pat. No. 5,585,481. The detection probe was provided to thereaction mixtures in both “cold” and “hot” forms, for the reasonsdescribed in Example 5, and the cold:hot probe ratio of each reactionmixture was 4000:1. As with the promoter and terminatingoligonucleotides, the cap had a 3′-terminal blocking moiety consistingof the 3′ to 3′ linkage described in Example 1.

The reaction mixtures were all template-free and tested in replicates ofthree, with a different set of reaction mixtures being prepared for eachpriming oligonucleotide. The amplification and detection steps werecarried out as in Example 2 using 30 pmol/reaction each of the promoterand priming oligonucleotides, 5 pmol/reaction of the terminatingoligonucleotide, 20 fmol/reaction of the hot probe, and 80 pmol/reactionof the cold probe. Signal from the tubes was measured in relative lightunits (“RLU”) and the averages of those RLU values are set forth inTable 9 below. The results indicate that the capped primingoligonucleotide inhibited primer-dependent side-product formation to agreater extent than did the looped priming oligonucleotides, althoughuse of the looped priming oligonucleotides resulted in lessprimer-dependent side-product formation than when the uncapped primingoligonucleotide was used in this amplification system.

TABLE 8 Inhibition of Primer-Dependent Side-product Formation UsingLooped Priming Oligonucleotides and Caps Priming Oligonucleotide Avg.RLU Uncapped 2,246,565 LPO V 1,497,699 LPO VII 1,040,960 Capped 106,134

Example 12 Comparison of RNA Transcript Production in the Presence andAbsence of Extender Oligonucleotides

This experiment examined the effect of extender oligonucleotides onamplicon production in amplification reaction mixtures containing ablocked promoter oligonucleotide. The extender oligonucleotides of thisexperiment were either blocked or unblocked and had the base sequence ofSEQ ID NO:55 cctccaggaccccccctcccgggagagccata. A 3′-end blockedterminating oligonucleotide was included that was made up of 2′-O-methylribonucleotides having the base sequence of SEQ ID NO:56auggcuagacgcuuucugcgugaaga. The target nucleic acid (“target”), primingoligonucleotide and promoter oligonucleotide were the same as those usedin Example 1. The blocking moiety of each blocked oligonucleotide usedin this experiment was a 3′-terminal blocking moiety consisting of the3′-to-3′ linkage described in Example 1. Cold and hot probes were usedfor detection of transcription products and had the sequence of SEQ IDNO:7. The hot probe of this experiment was identical to the firstdetection probe used in Example 5.

Six groups of amplification reaction mixtures were tested in replicatesof four as follows: (i) no extender oligonucleotide and no target (groupI); (ii) no extender oligonucleotide and 100 copies of target (groupII); (iii) blocked extender oligonucleotide and no target (group III);(iv) blocked extender oligonucleotide and 100 copies of target (groupIV); (v) unblocked extender oligonucleotide and no target (group V);(iv) unblocked extender oligonucleotide and 100 copies of target (groupVI). Reaction tubes from the six groups were set-up with 30 μLAmplification Reagent containing 6 pmol of the priming oligonucleotide,4 pmol of the promoter oligonucleotide and 0.8 pmol of the terminatingoligonucleotide. The reaction tubes of groups III and IV contained 4pmol of the blocked extender oligonucleotide, and the reaction tubes ofgroups V and VI contained 4 pmol of the unblocked extenderoligonucleotide. As indicated above, the reaction tubes of groups II, IVand VI further contained 100 copies of target, while those of groups I,III and V contained no target. The reaction mixtures were overlaid with200 μL Oil Reagent, and the tubes were then sealed and vortexed for 10seconds before being incubated in a 60° C. water bath for 10 minutes.The tubes were then transferred to a 41.5° C. water bath and incubatedfor 15 minutes before adding 10 μL Enzyme Reagent. After adding EnzymeReagent, the tubes were again sealed and hand-shaken horizontally for 5to 10 seconds to fully mix the components of the reaction mixtures. Thetubes were returned to the 41.5° C. water bath and incubated for anadditional 60 minutes to permit amplification of the target sequence.Following amplification, the tubes were removed from the 41.5° C. waterbath and placed in an ice water bath for two minutes.

Detection of RNA transcription products was performed essentially asdescribed in Example 2 (reaction tubes were vortexed rather thanhand-shaken) using 100 fmol/reaction of the hot probe and 300pmol/reaction of the cold probe. The averaged results are set forth inTable 9 in relative light units (“RLU”) and demonstrate that theextender oligonucleotides of this experiment contributed to faster ratesof amplification. The coefficient of variation values (“% CV”) appearingin Table 9 for the different reaction conditions tested constitute thestandard deviation of the replicates over the mean of the replicates asa percentage.

TABLE 9 Effect of Extender Oligonucleotides on Amplicon Production CopyExtender Number Oligonucleotide Avg. RLU % CV 0 None 4239 13 100 None70,100 28 0 Blocked 4721 30 100 Blocked 337,964 12 0 Unblocked 13,324 76100 Unblocked 869,861 12

Example 13 Amplification of a DNA Target Sequence

This experiment was conducted to determine the sensitivity of anamplification system according to the present invention for a targetregion contained within double-stranded DNA (“dsDNA”). The exemplarydsDNA used in this experiment was a cloned transcript derived from theorfX gene of a methicillin-resistant strain of Staphylococcus aureus.Included for amplification were a priming oligonucleotide, a displaceroligonucleotide, a terminating oligonucleotide and a promoteroligonucleotide. The priming oligonucleotide targeted a sequencecontained within the 3′-end of the target region and had the basesequence of SEQ ID NO:35; the displacer oligonucleotide targeted asequence present in a target nucleic acid containing the target region5′ to the target region and had the base sequence of SEQ ID NO:57cuugctcaattaacacaacccgcatc, where the underlined bases were LNAs; theterminating oligonucleotide targeted a sequence present in the targetnucleic acid 3′ to the target region and had the base sequence of SEQ IDNO:58 gcgttggttcaauuc, where the underlined bases were LNAs (theterminating oligonucleotide was not fully comprised of LNAs to limitfolding); and the promoter oligonucleotide had the base sequence of SEQID NO:34 aatttaatacgactcactatagggagaacgcatgacccaagggcaaagcgactttg, wherethe underlined portion was a T7 promoter sequence (SEQ ID NO:3) and theremainder was a hybridizing sequence (SEQ ID NO:33) that targeted thecomplement of a sequence contained within the 5′-end of the targetregion. The promoter oligonucleotide and the terminating oligonucleotideboth included a 3′-terminal blocking moiety consisting of the 3′-to-3′linkage described in Example 1. Detection was carried out in real-timewith a molecular torch detection probe having the base sequence of SEQID NO:59 ccgucauuggcggaucagacgg. The base sequence of this probe wascomprised of 2′-O-methylribonucleotides and nucleotides 17 and 18,reading 5′-to-3′, were joined to each other by a 9-carbon linker. Thedetection probe was synthesized to include interacting DABCYL and FAMlabels using 3′-DABCYL CPG (Prime Synthesis, Inc., Aston, Pa.; Cat. No.N-9756-10) and a 6-FAM phosphoramidite joined to the 5′-end (BioGenexInc., San Ramon, Calif.; Cat. No. BGX-3008-01). The probe was alsosynthesized to include a 9-carbon linker positioned between nucleotides17 and 18 reading 5′-to-3′ (Glen Research Corporation, Sterling, Va.;Cat. No. 10-1909-90). The synthesized probe was purified bypolyacrylamide gel electrophoresis and reverse phase HPLC. The targetbinding portion of the probe consisted of SEQ ID NO:37.

The amplification reactions of this experiment were performed inreplicates of six for each copy number and, for this, each of twelvetest wells of a microtiter plate was provided with 30 μL of anamplification reagent comprising 44.1 mM HEPES, 2.82% (w/v) trehalose,30.6 mM MgCl₂, 33 mM KCl, 0.3% (v/v) ethanol, 0.1% (w/v) methyl paraben,0.02% (w/v) propyl paraben, 9.41 mM rATP, 1.8 mM rCTP, 11.8 rGTP, 1.8 mMUTP, 0.47 mM dATP, 0.47 mM dCTP, 0.47 mM dGTP, 0.47 mM dTTP, 15 pmolpriming oligonucleotide, 8 pmol displacer oligonucleotide, and 0.5 pmolterminating oligonucleotide at pH 7.7. Each well also contained 0, 10,100, 1000 or 10,000 copies of the transcript. The plate was covered witha sealing card and incubated for 10 minutes at 95° C. in the equivalentof a DNA Engine Opticon® 2 Real-Time PCR Detection System (Bio-RadLaboratories, Hercules, Calif.; Cat. No. CFD3220) to denature thedouble-stranded transcript, and then for 5 minutes at 42° C. to promotehybridization of the priming oligonucleotide, the displaceroligonucleotide and the terminating oligonucleotide to the targetnucleic acid. After incubating, the plate was removed from the detectionsystem and 10 μL of an enzyme reagent was added to each test well toinitiate amplification. The enzyme reagent comprised 58 mM HEPES, 3.03%(w/v) trehalose, 50 mM N-acetyl-L-cysteine, 1.04 mM EDTA, 120 mM KCl,10% (w/v) TRITON® X-100, 20% (v/v) glycerol, 15 pmol promoteroligonucleotide, 8 pmol detection probe, 360 RTU/μL MMLV reversetranscriptase (“RT”), and 80 U/μL T7 RNA polymerase at pH 7.0, where oneRTU of RT activity incorporates 1 nmol of dT into a substrate in 20minutes at 37° C. and one U of T7 RNA polymerase activity produces 5fmol of RNA transcript in 20 minutes at 37° C. Following enzymeaddition, the plate was placed on a Thermomixer R (Eppendorf NorthAmerica, Inc., Westbury, N.Y.; Cat. No. 5355 29716) pre-warmed to 44°C., covered with a sealing card, and agitated for 30 seconds at 1400rpm. The plate was returned to the 42° C. detection system, where anamplification reaction monitored in real-time was carried out for 100minutes, with fluorescent readings being taken every 24 seconds.Detection depended upon conformational changes in the probes hybridizedto amplification products, thereby resulting in the emission ofdetectable, fluorescent signals.

The results of this experiment are illustrated graphically in FIG. 8,where time in minutes is plotted on the x-axis and relative fluorescentsignal is plotted on the y-axis. The data show that this amplificationsystem had a sensitivity of at least 1000 copies of transcript. None ofthe control samples (0 transcript) exhibited detectable amplification.

Example 14 Comparison of Systems for Amplifying a DNA Target Sequence

A set of experiments was conducted to compare the sensitivities ofvarious related amplification systems of the present invention for atarget region contained within double-stranded DNA. Like Example 13, theexemplary target nucleic acid used in this experiment was a clonedtranscript derived from the orfX gene of a methicillin-resistant strainof Staphylococcus aureus. The priming oligonucleotide used in theseexperiments had the base sequence of SEQ ID NO:36. The remainder of theoligonucleotides were the same as those of Example 13. Except for thefirst experiment, each of the experiments excluded one of the followingcomponents of Example 13: (i) a terminating oligonucleotide; (ii) adisplacer oligonucleotide; (iii) a priming oligonucleotide; and (iv)heat to denature the double-stranded target. The experiments were run inreplicates of twelve following the general protocol of Example 13. Thesets of replicates contained either 0 or 1000 copies of the transcript.

The results of this experiment are illustrated graphically in FIGS.9A-E, where, again, time in minutes is plotted on the x-axis andrelative fluorescent signal is plotted on the y-axis.

The results of these experiments show the following: (i) all replicatescontaining the target were positive when the amplification system wasnot modified (see FIG. 9A); (ii) all replicates containing target werepositive when the terminating oligonucleotide was excluded from thereaction mixture (see FIG. 9B); (iii) 6 out of 12 replicates containingthe target were positive when the displacer oligonucleotide was excludedfrom the reaction mixture (see FIG. 9C); (iv) 3 out of 12 replicatescontaining the target were positive when the priming oligonucleotide wasexcluded from the reaction mixture (see FIG. 9D); and (v) 4 out of 12replicates containing the target were positive when the double-strandedtarget was not exposed to heat-denaturing conditions (i.e., 95° C. heatstep) prior to enzyme addition (see FIG. 9E). None of the controlsamples (0 transcript) exhibited detectable amplification.

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. A method of synthesizing multiple copies of a target sequence, saidmethod comprising: treating a target nucleic acid comprising a DNAtarget sequence with a priming oligonucleotide which hybridizes to the3′-end of said target sequence such that a primer extension reaction canbe initiated therefrom, wherein said priming oligonucleotide does notcomprise RNA; extending said priming oligonucleotide in a primerextension reaction with a DNA polymerase to give a first DNA primerextension product, at least a portion of said first primer extensionproduct being complementary to said target sequence; treating saidtarget nucleic acid with a displacer oligonucleotide which hybridizes tosaid target nucleic acid upstream from said priming oligonucleotide suchthat a primer extension reaction can be initiated therefrom; extendingsaid displacer oligonucleotide in a primer extension reaction with saidDNA polymerase to give a second DNA primer extension product thatdisplaces said first primer extension product from said target nucleicacid; treating said first primer extension product with a promoteroligonucleotide comprising first and second regions, said first regioncomprising a base sequence which corresponds to a region at the 5′-endof said target sequence and which hybridizes to said first primerextension product to form a promoter oligonucleotide:first primerextension product hybrid, and said second region comprising a promoterfor an RNA polymerase and situated 5′ to said first region, wherein saidpromoter oligonucleotide is modified to prevent the initiation of DNAsynthesis therefrom; transcribing from said promoteroligonucleotide:first primer extension product hybrid multiple first RNAproducts complementary to at least a portion of said first primerextension product using an RNA polymerase which recognizes said promoterand initiates transcription therefrom, wherein the base sequences ofsaid first RNA products are substantially identical to the base sequenceof said target sequence, provided that if said first primer extensionproduct has a defined 3′-end, then said method further comprisestreating said target nucleic acid with a binding molecule which binds tosaid target nucleic acid adjacent to or near the 5′-end of said targetsequence.
 2. The method of claim 1, wherein any oligonucleotide providedin said method which comprises a promoter for an RNA polymerase ismodified to prevent the initiation of DNA synthesis therefrom.
 3. Themethod of claim 1, wherein said priming oligonucleotide consists ofdeoxynucleotides and/or analogs thereof.
 4. The method of claim 1,wherein said promoter oligonucleotide is modified to include a blockingmoiety situated at its 3′-terminus.
 5. The method of claim 4, whereinsaid blocking moiety of said promoter oligonucleotide comprises asubstituent selected from the group consisting of: a modifiednucleotide, a nucleotide or a nucleotide sequence having a 3′-to-5′orientation, a 3′ alkyl group, a 3′2′-dideoxynucleotide, a 3′cordycepin, a 3′ alkane-diol residue, a 3′ non-nucleotide moiety, anucleotide sequence non-complementary to said target sequence, a nucleicacid binding protein, and mixtures thereof.
 6. The method of claim 1,wherein said promoter oligonucleotide further comprises an insertionsequence positioned between or adjacent to said first and secondregions, and wherein the presence of said insertion sequence in saidpromoter oligonucleotide enhances the rate at which said RNA productsare formed.
 7. The method of claim 1 further comprising, prior toextending said priming oligonucleotide in a primer extension reaction,exposing a double-stranded complex comprising said target nucleic acidto conditions sufficient to denature said complex.
 8. The method ofclaim 7, wherein said conditions include heat.
 9. The method of claim 1,wherein said target nucleic acid is in a double-stranded complex, andwherein said method does not comprise, prior to extending said primingoligonucleotide in a primer extension reaction, exposing said complex toconditions sufficient to denature said complex.
 10. The method of claim1 further comprising: treating said first RNA products with said primingoligonucleotide to form a priming oligonucleotide:first RNA producthybrid such that a primer extension reaction can be initiated from saidpriming oligonucleotide; extending said priming oligonucleotide in aprimer extension reaction with said DNA polymerase to give a second DNAprimer extension product complementary to said first RNA product, saidsecond primer extension product having a 3′-end which is complementaryto the 5′-end of said first RNA product; separating said second primerextension product from said first RNA product using an enzyme whichselectively degrades said first RNA product; treating said second primerextension product with said promoter oligonucleotide to form a promoteroligonucleotide:second primer extension product hybrid; extending the3′-end of said second primer extension product in said promoteroligonucleotide:second primer extension product hybrid to add a sequencecomplementary to said second region of said promoter oligonucleotide;and transcribing from said promoter oligonucleotide:second primerextension product hybrid multiple second RNA products complementary tosaid second primer extension product using said RNA polymerase, whereinthe base sequences of said second RNA products are substantiallyidentical to the base sequence of said target sequence.
 11. The methodof claim 10, wherein said priming oligonucleotide is extended using areverse transcriptase having an RNAse H activity.
 12. The method ofclaim 10, wherein said enzyme has an RNAse H activity, and wherein saidenzyme is other than a reverse transcriptase.
 13. The method of claim 1,wherein the activity of said DNA polymerase in said method issubstantially limited to the formation of said first and second primerextension products.
 14. The method of claim 1, wherein a 5′-region ofsaid displacer oligonucleotide includes one or more modifications forincreasing the binding affinity of said displacer oligonucleotide forsaid target nucleic acid, and wherein said modifications do not preventsaid displacer oligonucleotide from being extended in a primer extensionreaction.
 15. The method of claim 14, wherein said modifications arespaced at least 15 bases from the 3′-terminus of said displaceroligonucleotide.
 16. The method of claim 14, wherein said modificationsare LNAs.
 17. The method of claim 1, wherein said displaceroligonucleotide hybridizes to said target nucleic acid such that the3′-terminal base of said displacer oligonucleotide is adjacent to the5′-terminal base of said priming oligonucleotide.
 18. The method ofclaim 1, wherein said displacer oligonucleotide hybridizes to saidtarget nucleic acid such that the 3′-terminal base of said displaceroligonucleotide is spaced from 5 to 35 bases from the 5′-terminal baseof said priming oligonucleotide.
 19. The method of claim 1, wherein saidtarget nucleic acid is treated with said priming oligonucleotide priorto treating said target nucleic acid with said displaceroligonucleotide.
 20. The method of claim 1, wherein said first primerextension product is formed prior to treating said target nucleic acidwith said displacer oligonucleotide.
 21. The method of claim 1, whereinsaid target nucleic acid is treated with said priming oligonucleotideand said displacer oligonucleotide prior to exposing said target nucleicacid to a DNA polymerase.
 22. The method of claim 1 further comprisingtreating the 3′-end of at least one of said priming oligonucleotide andsaid displacer oligonucleotide with one or more caps, each said capcomprising a base region complementary to at least 3 bases at the 3′-endof said priming oligonucleotide or said displacer oligonucleotide,wherein the 5′-terminal base of each said cap is complementary to the3′-terminal base of said priming oligonucleotide or said displaceroligonucleotide, and wherein each said cap is modified to prevent theinitiation of DNA synthesis therefrom.
 23. The method of claim 22,wherein each said cap is complementary to no more than 8 bases at the3′-end of said priming oligonucleotide or said displaceroligonucleotide.
 24. The method of claim 22, wherein each said capprevents non-specific hybridization between said priming oligonucleotideor said displacer oligonucleotide and said promoter oligonucleotide wheneach said cap is hybridized to said priming oligonucleotide or saiddisplacer oligonucleotide.
 25. The method of claim 22, wherein each saidcap is a capping oligonucleotide modified to include a blocking moietysituated at its 3′-terminus.
 26. The method of claim 22, wherein the3′-end of at least one of said caps is covalently attached to the 5′-endof said priming oligonucleotide or said displacer oligonucleotide, andwherein said at least one cap hybridizes to the 3′-end of said primingoligonucleotide or said displacer oligonucleotide by forming a loop. 27.The method of claim 26, wherein said at least one cap is joined to saidpriming oligonucleotide or said displacer oligonucleotide via a linkerregion.
 28. The method of claim 27, wherein said linker region comprisesat least 5 nucleotides.
 29. The method of claim 27, wherein said linkerregion comprises at least 5 abasic nucleotides.
 30. The method of claim1 further comprising: treating said target nucleic acid with saidbinding molecule; and extending the 3′-end of said first primerextension product in said promoter oligonucleotide:first primerextension product hybrid to add a sequence complementary to said secondregion of said promoter oligonucleotide.
 31. The method of claim 30,wherein said binding molecule comprises an oligonucleotide having ablocking moiety situated at its 3′-terminus to prevent the initiation ofDNA synthesis therefrom.
 32. The method of claim 31, wherein saidbinding molecule comprises LNAs.
 33. The method of claim 30, whereinsaid binding molecule is a terminating oligonucleotide.
 34. The methodof claim 33, wherein the 5′-end of said terminating oligonucleotide iscomplementary to at least two bases at the 5′-end of said first regionof said promoter oligonucleotide.
 35. The method of claim 33, whereinthe 5′-end of said terminating oligonucleotide is complementary to atleast three but no more than ten bases at the 5′-end of said firstregion of said promoter oligonucleotide.
 36. The method of claim 30,wherein said binding molecule is a modifying oligonucleotide.
 37. Themethod of claim 36, wherein said modifying oligonucleotide is adigestion oligonucleotide.
 38. The method of claim 1 further comprisingtreating said first primer extension product with an extenderoligonucleotide, said extender oligonucleotide hybridizing to a regionof said first primer extension product 3′ to said promoteroligonucleotide of said promoter oligonucleotide:first primer extensionproduct hybrid, such that an extender oligonucleotide:first primerextension product hybrid is formed.
 39. The method of claim 38, whereinsaid extender oligonucleotide further comprises a blocking moietysituated at its 3′ terminus to prevent the initiation of DNA synthesistherefrom.
 40. The method of claim 38, wherein said extenderoligonucleotide hybridizes to said first primer extension product suchthat the 5′-terminal base of said extender oligonucleotide is spacedwithin three nucleotides of the 3′-terminal base of said promoteroligonucleotide.
 41. The method of claim 38, wherein said extenderoligonucleotide hybridizes to said first primer extension productadjacent said promoter oligonucleotide.
 42. The method of claim 1further comprising determining the presence or amount of said multiplecopies of said target sequence.
 43. The method of claim 42, wherein thepresence or amount of said multiple copies of said target sequence isdetermined with a detection probe having a detectable label.
 44. Themethod of claim 43, wherein the presence or amount of said multiplecopies of said target sequence is determined after said first RNAproducts are transcribed.
 45. The method of claim 43, wherein thepresence or amount of said multiple copies of said target sequence isdetermined as said first RNA products are being transcribed.
 46. Themethod of claim 45, wherein said probe is a self-hybridizing probe andincludes a pair of interacting labels.
 47. The method of claim 1,wherein said method is carried out at a substantially constanttemperature.